EP1781257A2 - Pharmaceutical formulations containing microparticles or nanoparticles of a delivery agent - Google Patents

Abstract

This invention relates to microparticles and/or nanoparticles containing a delivery agent and /or an active agent. This invention also relates to pharmaceutical formulations and solid dosage forms, including controlled release solid dosage forms of active agent and a delivery agent

Description

PHARMACEUTICAL FORMULATIONS CONl¹AIMNG MICROPARTICLES OR NANOPARTICLES OF A DELIVERY AGENT

[1] This application claims the benefit of U.S. Provisional Application No. 60/612,810, filed September 23, 2004, and U.S. Provisional Application No. 60/601,258, filed August 13, 2004, both of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[2] This invention relates to pharmaceutical formulations and methods for preparing the same.

BACKGROUND OF THE INVENTION [3] There is a continuing need for improved oral delivery systems for drugs, such as insulin.

SUMMARY OF THE INVENTION

[4] The present invention relates to microparticles and/or nanoparticles for oral administration containing a delivery agent compound alone or a combination of a delivery agent compound and an active agent. Formulations containing these particles (and, for particles containing only a delivery agent compound, and an active agent) provide significantly greater bioavailability of the active agent with less variability than oral administration of a simple mixture of the delivery agent compound and active agent as a powder, tablet, or capsule. Without being bound by any particular theory, it is believed that in at least some embodiments, this improvement may be due to (1) the small size of the micro- or nano-particles which permits them to pass from the stomach, through the pylorus (which typically has a diameter of 1000-2000 μm), to the small intestine, where particle dissolution and delivery agent-mediated drug absorption is believed to best occur, and (2) the intimate contact between the delivery agent compound and active agent in the particles which ensures that the delivery agent compound is present with the active agent at the site of absorption. Because the micro- and nano-particles freely pass through the pylorus into the small intestine, unlike a conventional tablet or capsule which must first become dissolved into particles sufficiently small to do so, variations caused by tablet disintegration and gastric transit modulated by gastric motility are minimized.

[5] According to one embodiment, the particles comprising a delivery agent compound and an active agent have a median particle size less than about 900 or 1000 μm. For example, the median particle size can range from about 45 to about 850 μm, from about 45 to about 150 μm, from about 150 to about 250 μm, from about 250 to about 425 μm, from about 425 to about 850 μm, from about 100 to about 1000 nm, or from about 500 to about 1000 nm. According to another embodiment, the particles have a median particle size less than about 1 μm. In some embodiments, particles may be as small as about 1 nanometer and as large as about 999 micrometers. For example, the particles may have a median particle size of less than about 999 micrometers, from about 1 nanometer to about 999 micrometers, about 1 to about 999 micrometers, about 1 to about 999 nanometers, about 45 to about 850 micrometers, about 45 to about 150 micrometers, about 150 to about 250 micrometers, about 250 to about 425 micrometers, about 425 to about 850 micrometers, about 100 to about 1000 nanometers, or about 500 to about 1000 nanometers. [6] Another embodiment is a pharmaceutical formulation comprising a delivery agent compound and an active agent in which the delivery agent compound is in the form of particles. The particles can have a median particle size of less than about 999 micrometers, about 1 nanometer to about 999 micrometers, about 1 to about 999 nanometers, or about 7 to about 16 micrometers. Optionally, the active agent may also be in the form of particles. For example, the median particle size of the active agent particles may be less than about 999 micrometers, about 1 nanometer to about 999 micrometers, about 1 to about 999 micrometers, or about 1 to about 999 nanometers. According to one embodiment, the delivery agent particles and the active agent particles both have a median particle size of about 1 to about 999 micrometers. According to another embodiment, the delivery agent particles and the active agent particles both have a median particle size of about 1 to about 999 nanometers. - . [7] Yet another embodiment is a pharmaceutical formulation comprising a delivery agent and an active agent in which the active agent is in the form of particles having a median particle size of less than about 999 micrometers. According to one embodiment, the median particle size of the active agent particles is about 1 nanometer to about 999 micrometers, about 1 to about 999 micrometers, or about 1 to about 999 nanometers.

[8] The particles can be in the form of fine granules or micro-beads (e.g. , beads having a round/ball shape and a diameter of about 0.2 mm to about 2.0 mm). The micro- beads may be formed by compression. In one embodiment, the pharmaceutical formulation includes micro-beads containing a delivery agent compound, which are coated with an active agent, such as insulin or heparin. The micro-beads may have a diameter ranging from about 0.2 mm to 2.0 mm. [9] The particles may also include a mucoadhesive, such as a cellulose derivative

(e.g. , CMC sodium (available from Aqualon of Wilmington, DE)) or a polyacrylic acid (e.g., Carbopol™ available from B.F. Goodrich of Cleveland, OH). The mucoadhesive can (1) facilitate adhesion to mucosa (including in the gastrointestinal tract) thereby prolonging delivery agent-active agent contact with the mucosa, (2) stabilize and protect the active agent (e.g. , in the case of insulin), and (3) increase the permeability of biomembranes (including mucosa) thereby improving delivery and increasing bioavailability of the active agent.

[10] It has also been discovered that oral administration of insulin in conjunction with a delivery agent compound by solid oral dosage forms that do not degrade in the stomach, but do degrade in the intestine, provides significantly greater bioavailability of the insulin. Such solid oral dosage forms containing insulin or a different active agent provide greater bioavailability than forms that degrade in the stomach and forms that do not contain the delivery agent compound. Without being bound by any particular theory, it is believed that this improvement is due to the sensitivity of insulin and other active agents to degradation by enzymes or acid found in gastric fluid. Because the solid oral dosage forms do not degrade in the stomach, the insulin and other active agents are protected from degradation until they reach the intestine. [11] Another embodimentof the invention is a pharmaceutical formulation (such as a solid oral dosage form) comprising a therapeutically effective amount of an active agent and a delivery agent, where the pharmaceutical formulation has a disintegration time of about 250 seconds to about 650 seconds when orally administered. In another embodiment, the disintegration time is about 350 to about 550 seconds when orally administered. In yet another embodiment, the disintegration time is greater than 60 seconds when orally administered. In yet another embodiment, the disintegration time is greater than 400 seconds when orally administered. Disintegration time can be determined in water at 37 ± 2°C using the method described in USP <701>. Disintegration times may range from about 1 second to as much as about 24 hours, or more, depending on many factors including, but not limited to, the particular active agent(s), delivery agent compound(s), and excipients included in the pharmaceutical formulation.

[12] Another embodiment is a pharmaceutical formulation (such as a solid oral dosage form) comprising a therapeutically effective amount of an active agent and a delivery agent, where the solid oral dosage form does not substantially disintegrate or dissolve in the stomach, but does substantially disintegrate or dissolve in the intestine. In a preferred embodiment, the active agent is insulin. In another preferred embodiment, the active agent is an insulin derivative.

[13] In another embodiment, the pharmaceutical formulation is a solid oral dosage form which is covered with an enteric coating to retard disintegration in the stomach.

Enteric coatings include, but are not limited to, hydroxypropyl methylcelmlose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, cellulose acetate phthalate, poly(methacrylic acid-ethylacrylate), and poly(methacrylic acid-methyl methacrylate).

[14] In yet another embodiment, the pharmaceutical formulations may be formulated to erode from the surface of the dosage form, rather than disintegrate.

[15] The pharmaceutical formulations may include enzyme-inhibiting agents to prevent enzymatic degradation of active agents in the pharmaceutical formulation.

[16] In one embodiment, the delivery agent is a compound having the following structure or a salt thereof:

O R² O

2OH Ar C N R¹ C OH Formula A

wherein

Ar is phenyl or naphthyl;

Ar is optionally substituted with one or more of -OH, halogen, Ci-C* alkyl, C1-C4 alkenyl, C1-C4 alkoxy. or C1-C4 haloalkoxy;

R¹ is C3-C20 alkyl, G-C20 alkenyl, phenyl, naphthyl, (Ci-C 10 alkyl) phenyl, (C1-C10 alkenyl)phenyl, (C1-C10 alkyl) naphthyl, (Ci-Cio alkenyl) naphthyl, pheny 1(C 1-C10 alkyl), ρhenyl(Ci-C 10 alkenyl), naphthyl(Ci-Cio alkyl), or naphthyl(Ci-Cio alkenyl);

R¹ is optionally substituted with Ci to Gt alkyl, G- to Gt alkenyl, Ci to C4 alkoxy, C1-C4 haloalkoxy, -OH, -SH, -CO2R⁸, or any combination thereof;

R² is hydrogen, Ci to -C4 alkyl, or C2 to Ct alkenyl; and R¹ is optionally interrupted by oxygen, nitrogen, sulfur or any combination thereof. The term "2-OH-Ar" in formula A refers to a phenyl or naphthyl group having a hydroxyl group at the 2-position.

[17] According to one embodiment, the compounds are not substituted with an amino group in the position alpha to the acid group. [18] Preferably, Ar is substituted with a halogen.

[19] Preferably, R² is hydrogen.

[20] Preferably, R¹ is unsubstituted.

[21] Preferably, R¹ is not interrupted.

[22] Preferably, R¹ is Ci-io, C3-9, C3-7, C3, C7, or Cp alkyl. According to one embodiment, R¹ is not branched.

[23] Preferred delivery agent compounds include, but are not limited to, N-(8-[2- hydroxybenzoyl]amino)caprylic acid (the free acid of SNAC), N-(10-[2- hydroxybenzoyl]amino)decanoic acid (the free acid of SNAC), 4-[(2-hydroxy-4-chloro- benzoyl)-amino]butanoic acid (the free acid of 4-CNAB), and salts thereof, and solvates and hydrates thereof. The salt can be, for example, a sodium salt, such as a monosodium (i.e., SNAC, SNAD, or 4-CNAB) or disodium salt.

[24] In another embodiment, the delivery agent is a compound having the following structure or a salt thereof:

wherein

R¹, R², R³, and R⁴ are independently H, -OH₃ halogen, Ci-C₄ alkyl, C₂-C₄ alkenyl, C₁- C₄ alkoxy, -C(O)R⁸, -NO₂, -NR⁹R¹⁰, or -N⁺R⁹R¹⁰R¹¹ (R¹²)^";

R⁵ is H, -OH, -NO₂, halogen, -CF₃, -NR¹⁴R¹⁵, -N⁺R¹⁴R¹⁵R¹⁶ (R¹³)^", amide, Ci-C₁₂ alkoxy, C₁-C₁₂ alkyl, C₂-Ci₂ alkenyl, carbamate, carbonate, urea, or -C(O)R¹⁸;

R⁵ is optionally substituted with halogen, -OH₃ -SH, or -COOH;

R⁵ is optionally interrupted by O, N, S, or -C(O)-;

R⁵ is a Ci-Cn alkylene, C₂-Ci₂ alkenylene, or arylene;

R⁶ is optionally substituted with a C₁-C₄ alkyl, C₂-C4 alkenyl, C₁-C₄ alkoxy, -OH, - SH, halogen, -NH₂, or -CO₂R⁸;

R⁶ is optionally interrupted by O or N;

R⁷ is a bond or arylene;

R⁷ is optionally substituted with -OH, halogen, -C(O)CH₃, -NR¹⁰R¹¹, or -N⁺R¹⁰R¹¹R¹²

R⁸ is H₅ C₁-C₄ alkyl, C₂-C₄ alkenyl, Or -NH₂;

R⁹, R¹⁰, R¹¹, and R¹² are independently H or C₁-Ci₀ alkyl;

R¹³ is a halide, hydroxide, sulfate, tetrafluoroborate, or phosphate;

R¹⁴, R¹⁵, and R¹⁶ are independently H, Ci-C₁₀ alkyl, C₁-Ci₀ alkyl substituted with- COOH, C₂-Ci₂ alkenyl, C₂-C₁₂ alkenyl substituted with -COOH, or -C(O)R¹⁷; R¹⁷ is -OH, C₁-C₁₀ alkyl, or C₂-C₁₂ alkenyl; and R¹⁸ is H, Ci-C₆ alkyl, -OH, -NR¹⁴R¹⁵, Or N⁺R¹⁴R¹⁵R¹⁶ (R¹³)^". [25] In yet another embodiment, the delivery agent is a compound having the following structure or a salt thereof:

Formula C wherein

R¹, R², R³, R⁴ and R⁵ are independently H, -CN, -OH₅ -OCH₃, or halogen, at least one ofR¹, R², R³, R⁴ and R⁵ being -CN; and R⁶ is a Ci-C₁₂ linear or branched alkylene, alkenylene, arylene, alkyl(arylene) or aryl(alkylene).

[26] In yet another embodiment, the delivery agent is a compound having the following structure or a. salt thereof:

Formula D wherein each occurrence of X is hydrogen, halogen, hydroxyl, or C₁-Ca alkoxy,

R is substituted or unsubstituted C1-C3 alkylene or substituted or unsubstituted C₂-C₃ alkenylene, and n is an integer from 1 to 4.

[27] In yet another embodiment, the delivery agent is a compound having the following structure or a salt thereof:

Formula E wherein

X is halogen, and R is substituted or unsubstituted C₁-Cs alkylene or substituted or unsubstituted C₂-C₃ alkenylene.

[28] Preferred delivery agent compounds include but are not limited to, N-(8-[2- hydroxybenzoyl]-amino)caprylic acid, N-(10-[2-hydroxybenzoyl]-amino)decanoic acid, 8-(2- hydroxy-4-methoxybenzoylamino)octanoic acid, 8-(2-hydroxy-5-chlorobenzoylamino)- octanoic acid, 4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoic acid, and pharmaceutically acceptable salts thereof. The pharmaceutical formulations of the present invention may include any of the aforementioned delivery agent compounds, or any other delivery agent compounds, alone or in combination with one or more additional delivery agent compounds. [29] Suitable active agents include but are not limited to, proteins, polypeptides, peptides, hormones, polysaccharides, as well as synthetic, natural or recombinant sources thereof: growth hormones; growth hormone releasing hormones; growth hormone releasing factor, interferons; interleukin-1; interleukin-2; insulin, optionally having counter ions including zinc, sodium, calcium and ammonium; insulin-like growth factor; heparin; calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; protease inhibitors; adrenocorticotropic!, gonadotropin releasing hormone; oxytocin; leutinizing-hormone-releasing-hormone; follicle stimulating hormone; glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin; cromolyn sodium; vancomycin; desferoxamine; bisphosphonates; parathyroid hormone; anti-migraine agents; glucagon-like peptide 1 (GLP-I); antimicrobials; vitamins; and analogs, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these compounds; or any combination thereof. Preferred active agents include, but are not limited to, insulin and heparin (including, but not limited to, unfractionated heparin and low molecular weight heparin).

[30] In one embodiment of the present invention, the active agent is insulin. The insulin-containing pharmacuetical formulations of the present invention may also include a second hypoglycemic agent, an inhibitor of renal glucose reabsorption, or any combination of the foregoing (such as those described in U.S. Patent Publication No. 2005/0143424, which is hereby incorporated by reference). Suitable second hypoglycemic agents include, but are not limited to, insulin secretion-promoting agents, insulin resistance-ameliorating agents, insulin mimetics, α-glucosidase inhibitors, glucogenesis inhibitors, and any combination of any of the foregoing. According to one embodiment, the solid dosage form includes a sulfonyl urea, meglitinide analogue, biguanide (preferably metformin), or any combination of any of the foregoing. According to a preferred embodiment, the solid dosage form includes ' metformin. [31] Also provided is a pharmaceutical formulation, such as a solid dosage unit form, comprising the microparticles or nanoparticles of the present invention and/or having the disintegration times discussed above. The dosage unit form may be in the form of a tablet, capsule, powder, or sachet. The dosage unit form may have, alone or in combination, one or more enteric coatings, disintegrants, super disintegrants (such as sodium starch glycolate or croscarmellose sodium), and extra particle super disintegrants.

[32] In one embodiment, the solid oral dosage unit form is a fast disintegrating tablet. In another embodiment, the solid dosage unit form has a controlled or delayed release. [33] According to one embodiment, the present invention provides a tablet comprising the aforementioned particles and a disintegrant. In one embodiment, the disintegrant is a super disintegrant, such as sodium starch glycolate (Primojel^® available from Azebe UK Ltd. of South Humberside, UK), croscarmellose sodium (Primellose^® available from Azebe UK Ltd. of South Humberside, UK), or an extra particle super disintegrant.

[34] Another embodiment is a solid dosage form comprising a therapeutically effective amount of insulin and a delivery agent compound, where the solid dosage form has a disintegration time of at least 60 seconds when administered orally. The solid dosage form may have an enteric coating or be a surface eroding formulation. The solid dosage form may further comprise one or more enzyme inhibiting agents.

[35] Yet another embodiment is a solid dosage form comprising a therapeutically effective amount of insulin and a delivery agent compound, where the solid dosage form does not substantially disintegrate or dissolve in the stomach but does disintegrate or dissolve in the small intestine. The solid dosage form may have an enteric coating or be a surface eroding formulation. The solid dosage form may further comprise one or more enzyme inhibiting agents. [36] Another embodiment is a method for administering an active agent to an animal, particularly an animal in need of the active agent, by administering a pharmaceutical formulation comprising the microparticles or nanoparticles of the present invention and/or those having the disintegration times discussed above (i.e. those having a controlled or sustained release). Oral administration is a preferred route of administration. [37] Yet another embodiment is a method of treating a disease or for achieving a desired physiological effect in an animal by administering a pharmaceutical formulation of the present invention, including solid unit dosage forms comprising the microparticles or nanoparticles of the present invention and/or those having the disintegration times discussed above (i.e. those having a controlled or sustained release). Yet another embodiment is a method of increasing the oral bioavailability of active agents by orally administering a pharmaceutical formulation of the present invention.

[38] Yet another embodiment is a method of treating diabetes and/or reducing the incidence of systemic hyperinsulinemia associated with chronic dosing of insulin in a mammal (such as in a human, particularly a human in need thereof) by administering to the mammal a therapeutic effective amount of an insulin-containing pharmaceutical formulation of the present invention, e.g., those comprising the microparticles or nanoparticles of the present invention and/or those having the disintegration times discussed above. In one embodiment, the delivery agent compound is the free acid of 4-CNAB or a pharmaceutically acceptable salt thereof. The pharmaceutical formulation may be administered on a chronic basis.

[39] Yet another embodiment is a method of treating impaired glucose tolerance, early stage diabetes, or late stage diabetes or achieving glucose homeostasis in a mammal (such as in a human, particularly in need thereof) by administering to the mammal a therapeutic effective amount of an insulin-containing pharmaceutical formulation of the present invention, such as a pharmaceutical formulation comprising the microparticles or nanoparticles of the present invention and/or having the disintegration times discussed above. In one embodiment, the delivery agent compound is the free acid of 4-CNAB or a pharmaceutically acceptable salt thereof. The pharmaceutical formulation may be administered on a chronic basis.

[40] Yet another embodiment is a method of treating a human diabetic patient by orally administering to the human diabetic patient on a chronic basis a therapeutic effective amount of an insulin-containing pharmaceutical formulation described herein.

[41] Yet another embodiment is a method of preparing the micro- and nanoparticles of the present invention by drying a solution of a delivery agent compound and an active agent, for example, until a solid is formed, and optionally, isolating the particles. Preferably, the mixture is homogenous (e.g., the delivery agent compound and the active agent are uniformly distributed throughout the mixture). The method includes co-drying a mixture of the delivery agent compound, the active agent, and a solvent. Suitable solvents include, but are not limited to, hydroxylic solvents, water, and mixtures thereof. According to one embodiment, the mixture is dried at from about 10 to about 40° C (e.g., at room temperature). Preferably, the drying is performed at a controlled temperature. According to one embodiment, the drying is performed over an inert gas (preferably nitrogen gas). The dried material may optionally be milled and/or sieved to obtain the desired particle size. This method results in particles containing a homogeneous mixture of the delivery agent compound and the active agent.

[42] Another method of preparing the micro- and nano-particles of the present invention is by lyophilizing a mixture of the delivery agent compound, the active agent, and a solvent. Suitable solvents include, but are not limited to, hydroxylic solvents, water, and mixtures thereof.

[43] Yet another method of preparing the micro- and nano-particles of the present invention is by (1) dissolving a delivery agent compound and an active agent in a supercritical fluid, and (2) decreasing the system pressure to deposit the delivery agent compound and active agent as extremely fine particles. The deposition is a result of the rapid expansion of the supercritical solution.

[44] The following embodiments are collectively referred to herein as the "solid pharmaceutical composition embodiments" .

[45] The above features and many other attendant advantages of the invention will become better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS [46] Figure 1 depicts a schematic of direct dosing to the stomach and the jejunum.

[47] Figure 2 is a graph of the concentration of insulin level (±SEM) following direct dosing of coprocessed microparticles to the stomach and the jejunum over time.

[48] Figure 3 is a graph of the change in glucose level (±SEM) following direct dosing of coprocessed microparticles to the stomach and the jejunum over time.

[49] Figure 4 is a graph of the change in glucose (±SEM) following oral gavage from 3 different dosage forms: 1) a tablet made by compressing insulin and carrier, 2) a capsule containing microparticles of coprocessed insulin and carrier, and 3) a capsule containing a simple mixture of insulin and carrier, over time. [50] Figure 5 is a graph of the insulin level (±SEM) following oral gavage from

3 different dosage forms: 1) a tablet made by compressing insulin and carrier, 2) a capsule containing microparticles of coprocessed insulin and carrier, and 3) a capsule containing a simple mixture of insulin and carrier, over time.

[51] Figure 6 is a graph of the insulin level (±SEM) following oral gavage of a capsule containing microparticles of coprocessed insulin and carrier over time. Two of the ten rats exhibited significantly high insulin absorption. The average values with (N= 10) and without (N =8) inclusion of these two high responders are depicted in the graph.

[52] Figure 7 is a graph of the change in glucose (±SEM) following oral gavage of a capsule containing microparticles of coprocessed insulin and carrier over time. Two of the ten rats exhibited significantly high insulin absorption. The average values with (N- 10) and without (N =8) inclusion of these two high responders are depicted in the graph. [53] Figure 8 is a chart of the estimated absolute bioavailability (±SEM) from in situ dosing of coprocessed insulin and carrier to the stomach and the jejunum. Two compositions were evaluated: 1) insulin (0.25mg/kg) + delivery agent (37.5mg/kg), and 2) insulin (0.5mg/kg) + delivery agent (75mg/kg). [54] Figure 9 is a chart of the estimated absolute bioavailability of insulin level

(±SEM) from 1) subcutaneous administration, 2) direct dosing to the stomach, 3) direct dosing to the jejunum, 4) a tablet made by compressing insulin and carrier, 5) a capsule containing microparticles of coprocessed insulin and carrier with and without inclusion of the two high responders, and 6) a capsule containing a simple mixture of insulin and carrier.

[55] Figure 10 is a chart of the estimated bioavailability of insulin in the portal vein (±SEM) from 1) direct dosing to the stomach, 2) direct dosing to the jejunum, 3) a tablet made by compressing insulin and carrier, 4) a capsule containing microparticles of coprocessed insulin and carrier with and without inclusion of the two high responders, and 5) a capsule containing a simple mixture of insulin and carrier.

[56] Figure 11 is a chart of the estimated bioavailability (±SEM) of insulin relative to subcutaneous administration from 1) direct dosing to the stomach, 2) direct dosing to the jejunum, 3) a tablet made by compressing insulin and carrier, 4) a capsule containing microparticles of coprocessed insulin and carrier with and without inclusion of the two high responders, and 5) a capsule containing a simple mixture of insulin and carrier. [57] Figure 12 is a chart of the estimated bioavailability of insulin in the portal vein relative to subcutaneous administration (±SEM) from 1) direct dosing to the stomach, 2) direct dosing to the jejunum, 3) a tablet made by compressing insulin and carrier, 4) a capsule containing microparticles of coprocessed insulin and carrier with and without inclusion of the two high responders, and 5) a capsule containing a simple mixture of insulin and carrier.

[58] Figure 13 is a graph of the individual insulin levels following oral gavage of a capsule containing microparticles of coprocessed insulin and carrier over time. Rat 14 and rat 17 exhibited significantly high insulin absorption. The average values with (N= 10) and without (N =8) inclusion of these two high responders are depicted in the graph.

[59] Figure 14 is a graph of the individual glucose change following oral gavage of a capsule containing microparticles of coprocessed insulin and carrier over time. Rat 14 and rat 17 exhibited significantly high insulin absorption. The average values with (N= IO) and without (N =8) inclusion of these two high responders are depicted in the graph. . [60] Figure 15 is a graph of the individual insulin level following oral gavage of a capsule containing microparticles of coprocessed insulin and carrier over time. Rats 14 and 17 were omitted. The average value from N = 8 is depicted in the graph.

[61] Figure 16 is a graph of the individual glucose change following oral gavage of a capsule containing microparticles of coprocessed insulin and carrier over time. Rats 14 and 17 were omitted. The average value from N=8 is depicted in the graph. [62] Figure 17 is a graph depicting the changes over time in serum glucose levels in rhesus monkeys that have been fed formulations 1-6, described below, containing insulin and a delivery agent. These formulations have varying disintegration times.

[63] Figure 18 is a graph depicting the changes over time in serum insulin concentration rhesus monkeys that have been fed formulations 1-6, described below, containing insulin and a delivery agent. These formulations have varying disintegration times.

[64] Figure 19 is a graph of anti-factor Xa activity (U/ml) versus time in monkeys after administration of the SNAD/heparin formulation described in Example 10.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[65] The "particles," "micro-beads," and "granules" described herein may be any shape and can include one or more ingredients in addition to the delivery agent compound and/or active agent. The specific ingredients of any given particle, micro-bead, or granule, may also depend on the processes used and will not necessarily be the same in each individual particle, micro-bead, or granule from a batch.

[66] For example, where particles, micro-beads, or granules of an active agent are prepared separately from particles, micro-beads, or granules of a delivery agent compound, the active agent particles, micro-beads, or granules will, generally, not comprise delivery agent compound, and the delivery agent particles, micro-beads, or granules will, generally, not comprise active agent, though each particle, micro-bead, or granule may comprise other ingredients, as disclosed herein.

[67] In other embodiments, particles, micro-beads, or granules may be formed from a solution, suspension or mixture, in liquid or dry form, without limitation, which comprises at least an active agent and a delivery agent compound. Thus, for example, any given particle, micro-bead, or granule comprises both active agent and delivery agent compound, and may further comprise one or more other ingredients.

[68] The terms "diameter" and "median particle size" are generally used to refer to the dimensions of particles, micro-beads, and granules. The "median particle size" or "diameter" was determined as follows for Examples 8, 9, 10.

[69] Instrument: Mastersizer 2000 (EQ 202, model MS2K, serial number 34315-67)

[70] Manufacturer: MALVERN instruments, England

[71] Software: Mastersizer 2000 - . [72] Accessory: Scirocco 2000 (A) (model ADA 2000, serial number 34270/73)

[73] Dispersant: Dry dispersion

[74] Analysis model: General purpose

[75] Particle RI: 1.520 [76] Obscuration: 1 - 6%

[77] Standards: Malvern Quality Audit Standard for Sample Dispersion Units [78] The Malvern Mastersizer 2000 determines particle size by laser diffraction and model fitting. A well-dispersed sample in any two-phase system (e.g., powders, suspensions, or emulsions) is introduced into the path of a He-Ne laser focused with a lens of a length suitable for particle sizes present in the sample. The scattering pattern of particles in the laser path is measured by an array of detectors, with each detector measuring data from a particular range of angles.

[79] The Malvern apparatus assumes that the particles being measured are perfect spheres. For non-spherical particles the resulting particle size distribution may be different from those obtained by methods based on others principles. The electronic measurements will often have to be accompanied by microscopic investigation to determine the type of particles being investigated. For irregularly shaped particles, the particle size data obtained from Mastersizer 2000 will be interpreted as the diameter of an imaginary sphere that is equivalent in volume to the measured particle. (Note: d(0.1) is the size of particle for which 10% of the sample is below this size, d(0.5) is the size of particle for which 50% of the sample is below this size, and d(0.9) is the size of particle for which 90% of the sample is below this size.

[80] Generally, this apparatus measures one dimension of a, e.g. , particle as it travels past a laser; i.e., it measures the length of a straight line through the particle. For irregular particles, this results in a variation of results since the orientation of a particle relative to the laser may result in the single measurement being taken of that individual particle's longest, shortest, or any other dimension. However, a measurement is taken of a number of particles and a median diameter or size is calculated. Thus, "size" or "diameter" figures are estimates of the median "size" or "diameter" of particles. Alternatively, "diameter" or "size" was measured by a sieve method described in Example 1. "Diameter" should not be read to necessarily imply a spherical shape or a circular dimension, though in certain embodiments, e.g., particles may have rounded edges or generally spherical shapes.

[81] It should be understood, also, that the invention is not limited to particles, micro-beads, or granules which fall within a narrow range of "sizes" or "diameters". Thus, for example, some embodiments may comprise, depending at least on the ingredients and processes used, some particles which fall within, for example, both the nanometer and micrometer scale, in the same batch. The actual "sizes" or "diameters" of the individual particles may fall within a relatively narrow or relatively large range.

[82] As used herein and in the appended claims, the singular forms "a," "an," and "the," include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a particle" includes one or more of such particles, reference to "an" active agent includes one or more of such active agents, and "a" delivery agent includes one or more delivery agents,

[83] The term "about" generally means within 10%, preferably within 5% , and more preferably within 1 % of a given value or range.

[84] The term "hydrate" as used herein includes, but is not limited to, (i) a substance containing water combined in the molecular form and (ii) a crystalline substance containing one or more molecules of water of crystallization or a crystalline material containing free water. [85] The term "solvate" as used herein includes, but is not limited to, a molecular or ionic complex of molecules or ions of a solvent with molecules or ions of the delivery agent compound or salt thereof, or hydrate or solvate thereof.

[86] The term "delivery agent" refers to any of the delivery agent compounds disclosed herein. [87] The term "SNAC" refers to the monosodium salt of N-(8-[2- hydroxybenzoyl]-amino)caprylic acid, including the various polymorphic forms of the monosodium salt described in U.S. Provisional Application No. 60/569,476, filed May 6, 2004 (which is hereby incorporated by reference) unless otherwise indicated.

[88] The term "SNAD" refers to the monosodium salt of N-(10-[2- hydroxybenzoyl]-amino)decanoic acid, unless otherwise indicated. The term "disodium salt of SNAD" refers to the disodium salt of N-(10-[2-hydroxybenzoyl]-amino)decanoic acid.

[89] The term "5-CNAC" refers to the monosodium salt of N-(8-[2-hydroxy~5- chlorobenzoyl]-amino)octanoic acid, unless otherwise indicated.

[90] The term "4-CNAB" refers to the monosodium salt of sodium N-4-[(2- hydroxy-4-chlorobenzoyl)amino]butanoate, including anhydrous, monohydrate, and isopropanol solvates thereof and various polymorphic forms of the monosodium salt described in International Publication No. WO 03/057650 (which is hereby incorporated by reference), unless otherwise indicated.

[91] An "effective amount of active agent" is an amount of active agent which is effective to treat or prevent a condition in a living organism to whom it is administered over some period of time, e.g., provides a therapeutic effect during a desired dosing interval.

[92] The term "insulin" refers to all forms of insulin, including, but not limited to, naturally derived insulin and synthetic forms of insulin, such as those described in U.S. Patent Nos. 4,421,685, 5,474,978, and 5,534,488, each of which is hereby incorporated by reference in its entirety.

[93] The term "insulin derivatives" refers to insulin-derived proteins and peptides with insulin actions, and include, for example, lispro, BlOAsp and HOE-901.

[94] An "effective amount of delivery agent" is an amount of the delivery agent which enables and/or facilitates the absorption of a desired amount of active agent via any route of administration (such as those discussed in this application including, but not limited to, the oral (e.g. , across a biological membrane in the gastrointestinal tract), nasal, pulmonary, dermal, buccal, vaginal, and/or ocular route).

[95] The terms "alkyl" and "alkenyl" as used herein include linear and branched alkyl and alkenyl substituents, respectively. [96] The phrase "pharmaceutically acceptable" refers to additives or compositions that are physiologically tolerable when administered to a mammal. [97] The phrase "substantially disintegrate" means that about 75% to about 95% of the total volume of the tablet will break apart and dissolve into its component parts (e.g. insoluble coated particles, insoluble disintegrant, etc.), and the tablet is no longer intact except for small aggregates. [98] "Surface eroding formulation" refers to formulations that do not disintegrate but instead erode, e.g., the formulation dissolves from the surface over a pre-determined period of time and the tablet generally remains intact and retains its overall shape. The surface eroding formulations allow for sustained release of an active agent over the predetermined time period. [99] The terms "micronize" and "micronized" generally refer to a process, or particles which have been processed, such that their diameters/sizes are within the general range of microparticles and/or nanoparticles.

[100] The term "microparticle" generally includes particles having a diameter ranging from about 1 to about 999 micrometers (microns, μm). ■ . [101] The term "nanoparticle" generally includes particles having a diameter ranging from about 1 to about 999 nanometers (nm)..

[102] The term "insulin derivatives" includes insulin-derived proteins and peptides with insulin actions, and include, for example, lispro, BlOAsp and HOE-901.

[103] "Insulin secretion-promoting agents" exert their hypoglycemic action, by mainly influencing pancreatic β-cells to promote insulin secretion into blood, and include, for example, sulfonylureas (for example, tolbutamide, chlorpropamide, glibenclamide (glyburide), glipizide, glimeperide, and gliclazide); and meglitinide analogues (for example, repaglinide, nateglinide, meglitinide and mitiglinide (KAD-1229))). Other insulin secretion-promoting agents are, for example, K⁺-ATP channel inhibitors (for example, BTS-67-582), glucagon-like peptide-1 receptor agonists (for example, glucagon-like peptide- 1, exendin-4 and NN-2211) and dipeptidyl peptidase-IV inhibitors with an effect of enhancing the action of glucagon-like peptide-1. According one embodiment, the insulin secretion-promoting agent is a sulfonylurea or meglitinide analogue.

[104] The term "insulin resistance-ameliorating agents" includes agents exerting hypoglycemic action by enhancing the action of insulin in target tissues, and include for example peroxisome proliferator activator receptor (PPAR)-γ agonists (for example, thiazolidine-based compounds such as pioglitazone, rosiglitazone, and ciglitazone; or non- thiazolidine-based compounds such as GI-262570, JTT-501, YM-440, NN-622 and KRP- 297), PPAR-γ antagonists and protein tyrosine phosphatase inhibitors. The insulin resistance-ameliorating agents include, for example, pharmaceutical agents with a function ameliorating insulin resistance, for example biguanides (for example, metformin, phenformin and buformin, preferably metformin), PPAR-α agonists (fibrate-series compounds such as simfϊbrate, clofibrate, bezafibrate and clinofibrate and non-fibrate-series compounds), anti-obesity agents (for example, 5-hydroxytryptamine (5-HT) reuptake inhibitors such as sibutramine, lipase inhibitors such as orlistat and adrenalin β-receptor agonists such as AJ-9677). Preferred insulin resistance-ameliorating agents include, but are not limited to, biguanides, such as metformin.

[105] The term "insulin mimetics" refers to agents expressing the hypoglycemic action through physiological insulin action, namely the action promoting glucose uptake into cells, in a manner more or less independent to insulin, except for insulin derivatives, and include for example insulin receptor-activating agents (for example, CLX-0901 and L-

783281) and vanadium.

[106] The term "α-glucosidase inhibitors" refers to agents expressing the hypoglycemic action through suppression of glucose absorption into bodies, mainly via the inhibition of α-glucosidase in the intestinal tube and include, for example, acarbose, voglibose and miglitol.

[107] The term "glucogenesis inhibitors" refers to agents expressing hypoglycemic action mainly through the inhibition of glucogenesis, and include for example glucagon secretion suppressors (for example, M&B-39890A and octreotide), fatty acid decomposition inhibitors (for example, nicotinic acid derivatives and carnitine palmitoyltransferase- 1 inhibitor) and ghicose-6-phosphatase inhibitors.

[108] The term "inhibitor of renal glucose reabsorption" refers to agents which inhibit glucose reabsorption in uriniferous tubules. The primary action of the inhibitor of renal glucose reabsorption is not involved in the promotion of the uptake into target tissue cells, the suppression of the absorption from intestinal tube, or the hypoglycemic action via the suppression of the synthesis in tissues. Suitable inhibitors of renal glucose reabsorption include, but are not limited to, those described in U.S. Patent Publication No.

2005/0143424, which is hereby incorporated by reference.

Delivery Agent Compounds [109] The delivery agent compound may be any of those described in U.S. Patent Nos. 5,650,386 and 5,866,536 and International Publication Nos. WO94/23767, WO95/11690, W095/28920, WO95/28838, W096/10396, W096/09813, WO96/12473, WO96/12475, WO96/30036, WO96/33699, WO97/31938, WO97/36480, WO98/21951, WO98/25589, W098/34632, W098/49135, WO99/16427, WO00/06534, WOOO/07979, WO00/40203, WO00/46182, WOOO/47188, WOOO/48589, WOOO/50386, WOOO/59863, WOOO/59480, WO01/32130, WO01/32596, WO01/34114, WO01/44199, WO01/51454, WO01/70219, WO01/92206, WO02/02509, WO02/15959, WO02/16309, WO02/20466, WO02/19969, WO02/070438, WO03/026582, WO02/100338, WO03/045306, WO03/26582, and WO 03/057170, all of which are hereby incorporated by reference.

[110] Non-limiting examples of delivery agent compounds include N-(8-[2- hydroxybenzoyl]amino)caprylic acid, N-(10-[2-hydroxybenzoyl]amino)decanoic acid, 8-(2- hydroxy-4-methoxybenzoylamino)octanoic acid, 8-(2-hydroxy-5-chlorobenzoyl- amino)octanoic acid, 4-[(2-hydroxy-4-chlorobenzoyl)amino]butanoic acid, and salts thereof. Preferred salts include, but are not limited to, monosodium and disodium salts.

[111] According to one embodiment, the delivery agent compound is N-(8-[2- hydroxybenzoyl]amino)caprylic acid or a pharmaceutically acceptable salt thereof.

. [112] According to another embodiment, the delivery agent compound is N-(10-[2- hydroxybenzoyl]amino)decanoic acid or a pharmaceutically acceptable salt thereof. [113] According to another embodiment, the delivery agent compound is 4-[(2- hydroxy-4-chlorobenzoyl)amino]butanoic acid or a pharmaceutically acceptable salt thereof.

[114] According to another embodiment, the delivery agent compound is 8-(2- hydroxy-5-chlorobenzoylamino)octanoic acid or a pharmaceutically acceptable salt thereof. [115] The delivery agent compounds may be in the form of the carboxylic acid or pharmaceutically acceptable salts thereof, such as sodium salts, and hydrates and solvates thereof. The salts may be mono- or multi-valent salts, such as monosodium salts and disodium salts (e.g., the disodium salt of 8-(2-hydroxy-5-chlorobenzoylamino)-octanoic acid, the disodium salt of N-(8-[2-hydroxybenzoyl]amino)caprylic acid, the disodium salt of N-(10-[2-hydroxybenzoyl]amino)decanoic acid). See, for example, International Publication No. WO 00/59863, which is hereby incorporated by reference The delivery agent compounds may contain different counter ions chosen for example due to their effect on modifying the dissolution profile of the carrier. [116] The delivery agent compounds may be prepared by methods known in the art, such as those discussed in the aforementioned publications (e.g., International Publication Nos. WO 98/34632, WO 00/07979, WO 01/44199, WO 01/32596, WO 02/02509, WO 02/20466, and WO 03/045306). SNAC, SNAD, 4-CNAB, and the free acid and other salts thereof may be prepared by methods known in the art, such as those described in U.S. Patent Nos. 5,650,386 and 5,866,536 and International Publication No. WO 02/02509, each of which are hereby incorporated by reference.

[117] Salts of the delivery agent compounds of the present invention may be prepared by methods known in the art. For example, sodium salts may be prepared by dissolving the delivery agent compound in ethanol and adding aqueous sodium hydroxide. [118] The delivery agent compound may be purified by recrystallization or by fractionation on one or more solid chromatographic supports, alone or linked in tandem. Suitable recrystallization solvent systems include, but are not limited to, acetonitrile, methanol, and tetrahydrofixran. Fractionation may be performed on a suitable chromatographic support such as alumina, using methanol/n-propanol mixtures as the mobile phase; reverse phase chromatography using trifluoroacetic acid/acetonitrile mixtures as the mobile phase; and ion exchange chromatography using water or an appropriate buffer as the mobile phase. When anion exchange chromatography is performed, preferably a 0- 500 mM sodium chloride gradient is employed.

[119] The delivery agent may contain a polymer conjugated to it by a linkage group selected from the group consisting Of -NHC(O)NH-, -C(O)NH-, -NHC(O), -OOC-, - COO-, -NHC(O)O-, -OC(O)NH-, -CH₂NH -NHCH₂-, -CHZNHC(O)O-, -OC(O)NHCH₂-,- CH2NHCOCH2O-, -OCH₂C(O)NHCH₂-, - NHC(O)CH₂O-, -OCH₂C(O)NH-, -NH-, -0-, and carbon-carbon bond, with the proviso that the polymeric delivery agent is not a polypeptide or polyamino acid. The polymer may be any polymer including, but not limited to, alternating copolymers, block copolymers and random copolymers, which are safe for use in mammals. Preferred polymers include, but are not limited to, polyethylene; polyacrylates; polymethacrylates; poly(oxyethylene); poly(propylene); polypropylene glycol; polyethylene glycol (PEG); and derivatives thereof and combinations thereof. The molecular weight of the polymer typically ranges from about 100 to about 200,000 daltons. The molecular weight of the polymer preferably ranges from about 200 to about 10,000 daltons. In one embodiment, the molecular weight of the polymer ranges from about 200 to about 600 daltons and more preferably ranges from about 300 to about 550 daltons.

Active Agents [120] Active agents suitable for use in the present invention include biologically active agents and chemically active agents, including, but not limited to, pesticides, pharmacological agents, and therapeutic agents. Suitable active agents include those that are rendered less effective, ineffective or are destroyed in the gastro-intestinal tract by acid hydrolysis, enzymes and the like. Also included as suitable active agents are those macromolecular agents whose physiochemical characteristics, such as, size, structure or charge, prohibit or impede absorption when dosed orally.

[121] For example, biologically or chemically active agents suitable for use in the present invention include, but are not limited to, proteins; polypeptides; peptides; hormones; polysaccharides, and particularly mixtures of muco-polysaccharides; carbohydrates; lipids; small polar organic molecules (i.e. polar organic molecules having a molecular weight of 500 daltons or less); other organic compounds; and particularly compounds which by themselves do not pass (or which pass only a fraction of the administered dose) through the gastro-intestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastro-intestinal tract; or any combination thereof.

[122] Further examples include, but are not limited to, the following, including synthetic, natural or recombinant sources thereof: growth hormones, including human growth hormones (hGH), recombinant human growth hormones (rhGH), bovine growth hormones, and porcine growth hormones; growth hormone releasing hormones; growth hormone releasing factor, interferons, including α (e.g., interferon alfacon-1 (available as Infergen^® from InterMune, Inc. of Brisbane, CA)), β and D; interleukin-1; interleukin-2; insulin, including porcine, bovine, human, and human recombinant, optionally having counter ions including zinc, sodium, calcium and ammonium; insulin-like growth factor, including IGF-I; heparin, including unfractionated heparin, heparinoids, dermatans, chondroitins, low molecular weight heparin, very low molecular weight heparin and ultra low molecular weight heparin; calcitonin, including salmon, eel, porcine and human; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; protease inhibitors; adrenocorticotropin, gonadotropin releasing hormone; oxytocin; leutinizing-hormone-releasing-hormone; follicle stimulating hormone; glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin; cromolyn sodium (sodium or disodium chromoglycate); vancomycin; desferoxamine (DFO); bisphosphonates, including alendronate, tiludronate, etidronate, clodronate, pamidronate, olpadronate, and incadronate; parathyroid hormone (PTH), including its fragments; antimigraine agents such as BIBN-4096BS and other calcitonin gene-related proteins antagonists; glucagon-like peptide 1 (GLP-I); antimicrobials, including antibiotics, anti- bacterials and anti-fungal agents; vitamins; analogs, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these compounds; or any combination thereof. Non- limiting examples of antibiotics include gram-positive acting, bacteriocidal, lipopeptidal and cyclic peptidal antibiotics, such as daptomycin and analogs thereof.

[123] According to one embodiment, the active agent is insulin.

[124] According to another embodiment, the active agent is heparin, such as unfractionated heparin or low molecular weight heparin.

[125] The amount of active agent used in a pharmaceutical composition or dosage unit form of the present invention is an amount effective to treat the target indication. However, the amount can be less than that amount when the composition is used in a dosage unit form because the dosage unit form may contain a plurality of delivery agent compound/ active agent, such compositions may contain a divided effective amount. The total effective amount can then be administered in cumulative units containing, in total, an effective amount of active agent. Moreover, those skilled in the field will recognize that an effective amount of active agent will vary with many factors including the age and weight of the animal, the animal's physical condition, as well as other factors.

[126] The total amount of active agent to be used of can be determined by methods known to those skilled in the art. However, because the compositions of the invention may deliver active agent more efficiently than compositions containing the active agent without the delivery agent, lower amounts of active agent than those used in prior dosage unit forms or delivery systems can be administered to the subject, while still achieving the same blood levels and/or therapeutic effects.

[127] According to one embodiment, insulin is administered at a dose of about 0.025 to about 1.0 mg per kilogram of body weight of the recipient per day (mg/kg/day), about 0.06 to about 0.25 mg/kg/day, or about 0.09 to about 0.19 mg/kg/day (based on the weight of active agent). The desired dose may be administered either as a single or divided dose.

[128] Generally an effective amount of delivery agent to facilitate the delivery of the active agent is administered with the active agent. According to one embodiment, the amount of delivery agent to active agent on a molar basis ranges from about 100:1 to about 1:1, from about 80:1 to about 2:1, or from about 20:1 to about 10:1. Delivery agent to active agent molar basis ranges may be higher than 100:1 for particular combinations of delivery agents and active agents. Alternatively, delivery agent to active agent ranges may be about 1:1 or lower, such as, e.g., 0.1:1 or lower, with particular combinations of delivery agents and active agents. [129] Dosage unit forms can also include any one or combination of excipients, disintegrants, lubricants, plasticizers, colorants, flavorants, taste-masking agents, sugars, sweeteners, and salts.

[130] The compositions of the subject invention are useful for administering biologically or chemically active agents to any animals, including but not limited to birds such as chickens, insects, fish, reptiles, mammals (including, but not limited to, rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and preferably humans).

[131] Another embodiment of the present invention is a method for the treatment or prevention of a disease or for achieving a desired physiological effect, such as those listed in the table 1 below, in an animal by administering the particles of the present invention. Preferably, an effective amount of the particles for the treatment or prevention of the desired disease or for achieving the desired physiological effect is administered.

Specific indications for active agents can be found in the Physicians' Desk Reference (58^th

Ed., 2004, Medical Economics Company, Inc., Montvale, NJ), which is herein incorporated by reference. The active agents in the table below include their analogs, fragments, mimetics, and polyethylene glycol-modified derivatives.

Table 1

Controlled or Sustained Release Formulations

[132] The solid dosage forms of the present invention may be formulated so as to prevent or retard break down in the stomach. Controlled release formulations suitable for use in the present invention may, for example, include an enteric coating or may be formulated to erode from the surface. [133] According to one embodiment, the solid oral dosage forms comprises a therapeutically effective amount of an active agent and a delivery agent, wherein the solid oral dosage form has a disintegration time of about 250 seconds to about 650 seconds when orally administered. In another embodiment, the disintegration time is about 350 to about 550 seconds when orally administered. In one embodiment the disintegration time is greater than 60 seconds when orally administered. In another embodiment, the disintegration time is greater than 400 seconds when orally administered. Disintegration time can be determined in water at 37 ± 2°C using the method described in USP <701>. [134] The solid dosage forms of the present invention may be covered by an enteric coating. The enteric coating may serve as the primary control for delaying the release of the drug composition or compositions in the solid dosage form. The enteric coating stays intact in the stomach and prevents or retards release into the stomach in the solid dosage form. Release of the active agent is delayed until the solid dosage form reaches the intestine. Once in the intestine, the higher pH causes release of the active agent. Enteric coatings include, but are not limited to, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, cellulose acetate phthalate, poly(methacrylic acid- ethylacrylate), and poly(methacrylic acid-methyl methacrylate). Other enteric coatings which may be used in accordance with the present invention are described in U.S. Patent No. 5,851,579, which is hereby incorporated by reference.

[135] In one embodiment of the present invention, the enteric coating is applied to the entire tablet, or other dosage form. In one embodiment the enteric coating is applied to a multi-particulate system, such as a system comprising microparticles and/or nanoparticles discussed above.

[136] The solid dosage forms of the present invention may be formulated to erode from the surface of the tablet (or other dosage uniform), or at the surface of the multi- particulate system (e.g. a system comprising microparticles discussed above). These surface erosion formulations slowly dissolve from the surface rather than disintegrate. By controlling the rate of surface erosion, release of the active agent and drug composition of the solid dosage form can be delayed. The surface erosion formulations can be formulated such that substantial release of the active agents or drug compositions do not occur until the solid oral dosage form reaches the intestines.

Enzyme Inhibiting Agents

[137] The solid dosage forms of the present invention (comprising the microparticles or nanoparticles of the present invention and/or having the disintegration times discussed above) may also include enzyme inhibiting agents. Enzyme inhibiting agents incorporated into the solid dosage unit forms may prevent the breakdown of insulin or other active agents that may be sensitive to enzymatic degradation. Enzyme inhibiting agents are described in U.S. Patent No. 6,458,383 which is hereby incorporated by reference. [138] Generally, inhibitory agents can be divided into the following classes: inhibitors that are not based on amino acids, including P-aminobenzamidine, FK-448, camostat mesylate and sodium glycocholate; amino acids and modified amino acids, including aπώioboronic acid derivatives and n-acetylcysteine; peptides and modified peptides, including bacitracin, phosphinic acid dipeptide derivatives, pepstatin, antipain, leupeptin, chymostatin, elastatin, bestatin, hosphoramindon, puromycin, cytochalasin potatocarboxy peptidase inhibitor, and amastatin; polypeptide protease inhibitors, including aprotinin (bovine pancreatic trypsin inhibitor), Bowman-Birk inhibitor and soybean trypsin inhibitor, chicken egg white trypsin inhibitor, chicken ovoinhibitor, and human pancreatic trypsin inhibitor; complexing agents, including EDTA, EGTA, 1,10-phenanthroline and hydroxychinoline; and mucoadhesive polymers and polymer-inhibitor conjugates, including polyacrylate derivatives, chitosan, cellulosics, chitosan-EDTA, chitosan-EDTA-antipain, polyacrylic acid-bacitracin, carboxymethyl cellulose-pepstatin, polyacrylic acid-Bowman- Birk inhibitor.

[139] The choice and levels of the enzyme inhibitor are based on toxicity, specificity of the proteases and the potency of inhibition, and will be apparent to those skilled in the art. - , [140] Without wishing to be bound by theory, it is believed that an inhibitor can function solely or in combination as: a competitive inhibitor, by binding at the substrate binding site of the enzyme, thereby preventing the access to the substrate (examples of inhibitors believed to operate by this mechanism are antipain, elastatinal and the Bowman Birk inhibitor); a non-competitive inhibitor that can be simultaneously bound to the enzyme site along with the substrate, as their binding sites are not identical; and/or a complexing agent due to loss in enzymatic activity caused by deprivation of essential metal ions out of the enzyme structure. Examples

[141] The following examples illustrate the invention without limitation. All parts are given by weight unless otherwise indicated.

Example 1 1. Test Articles a. Co-processed insulin/delivery agent microparticles used for site specific, in situ experiment and oral gavage experiments [142] Recombinant human zinc insulin (50 mg) and sodium 4-CNAB (7.5 g) were dissolved in 50 ml of deionized water. The clear solution was dried with nitrogen flow at room temperature for 24 hours. The obtained coprocessed cake was milled into fine particles, which were then sieved through a 40/60 mesh screen to obtain microparticles of a specific size range. The size of the microparticles used in the current study ranged from 250 to 420 μm. These microparticles contained by weight 0.55% of insulin, 9.5% of water and 89.5% of delivery agent. A total of approximately 90% (w/w) of insulin was recovered from this process.

[143] Particles were measured by passing them through seives with different size openings (850 μra, 425 μm, 250 μm, 150 μm, 45 μm). With this method, it can be determined that the median particle size ranges from about 45 to about 850 μm, from about 45 to about 150 μm, from about 150 to about 250 μm, from about 250 to about 425 μm, or from about 425 to about 850 μm. [144] Insulin content in the microparticles was measured with reversed phase HPLC (Phenomenex column: Luna 3u C 18 (2) 10OA, 150 x 4.6 mm, 3 micro; mobile phases: A, 0.1 % TFA in water; B, 0.1 % TFA in acetonitrile; Detector: UV280 ran). Water contents of the particles were measured with a 737 KF coulometer. b. Capsules loaded with the microparticles for oral gavage

[145] Gelatin capsules (size #9) were used in the rat studies. The necessary amount of microparticles loaded manually into the gelatin capsules were determined based on an average rat body weight of 350 mg. Each loaded capsule contained approximately 16 mg of the microparticles (equivalent to 0.0875 mg of insulin). c. Insulin/Delivery Agent mini-tablets for oral gavage experiment

[146] Insulin was well mixed with delivery agent at a ratio of 1:150 (w/w), which corresponded to 0.67% (w/w) of insulin. Based on an average rat body weight of 350 mg, a total amount of 26.43 mg of the mixed powder, which contained 0.175 mg of insulin and 26.26 mg of delivery agent, was directly compressed into tablets under a pressure of 1000 ps.i in a Carver press. The cylindrical mini-tablets were 2 mm in diameter and 6 mm in height. d. Capsules loaded with insulin/delivery agent physical blend for oral gavage experiment

[147] Insulin was well mixed with delivery agent at a ratio of 1: 150 (w/w). The amount of insulin and delivery agent mixture loaded manually into the gelatin capsules (size #9) were determined based on an average rat body weight of 350 mg. Each capsule contained 26.43 mg of the mixture (equivalent to 0.175 mg insulin). 2. Direct Dosing Procedures for In Situ Experiments

[148] A schematic of the direct dosing procedure is shown in Figure 1. Surgery was carried out in a clean environment using a clean lab coat, mask, safety goggle, gloves and surgical cap. Anesthesia was induced to the Sprague Dawley rats with 5% isoflurane as an induction concentration, and maintained at 2% isoflurane in pure oxygen to the completion of the study. a. Stomach direct dosing

[149] After the right jugular vein was catheterized for sampling blood, the skin over the esophagus and trachea was dissected, and the musculus digastricul venter rostralis (protective muscular bundles) was identified and dissected to make an access toward the esophagus. The esophagus was partially severed, and inserted with a 12 cm PE204 tubing for a segment of the esophagus measuring- 6-9 cm. The dosing formulation was introduced through this tubing using a blunt wire to push in the microparticles. After dosing, the esophagus was ligated with a 3-0 silk suture for preventing any leakage from the stomach. b. Jejunum direct dosing

[150] After the right jugular catheterization for the blood sampling, the abdominal cavity was opened by dissecting the linea alba toward the sternum, thus exposing the xiphoid cartilage. The most proximal segment of jejunum was first identified. A less vascularized section of the proximal jejunum was partially nipped, and a dosing tube was introduced toward the distal end. After dosing, the dosing tube was removed, and a 2 cm PE206 tubing was pushed in, and placed so that the nipped wound was located in the middle of both ends of the 2 cm tubing. A suture was tied around the tubing with jejunum at both ends, and the wound was closed with a drop of a vetbond™ tissue adhesive (available from 3M of St. Paul, Minnesota).

3. Oral Gavage Procedures

[151] Studies were carried out in Sprague Dawley rats (body weight was approximately 350 grams) by oral gavage administration. The mini tablets or capsules were administrated orally in rats using a modified gavage tubing with a trocar. Rats were fasted for about 24 hours and anesthetized by intramuscular administration of ketamine (44 mg/kg) and thorazine (1.5 mg/kg). At pre-determined time intervals, blood samples were drawn from the tail artery and were appropriately prepared as either plasma or serum for glucose and insulin bioassays. The animal was sacrificed at the end of the experiment and rat gastrointestinal mucosa was observed for any sign of local toxicity.

4. Bioassay Procedures

[152] Rat serum concentrations of insulin were determined using Insulin ELISA Test Kit (DSL Inc.). The limit of quantitation (LOQ) has been established at 12.5 DU/mL, w-i]th the calibrated linear range of the assay up to 250 QU/mL. Changes in blood glucose levels were measured using a glucometer.

5. Results a. Site Specific Study (In Situ) Results

[153] The concentration of insulin and the change in glucose level following direct dosing of the coprocessed microparticles to the stomach and the jejunum are shown in Figures 2 and 3, respectively. The individual data are listed in Tables 2 to 5. [154] Insulin concentration from dosing to the jejunum reached a maximum value at the first sampling point (w < 15 min) from each formulation. The corresponding faun of glucose occurred approximately 30 min. later.

Table 2

Direct dosing of coprocessed microparticles to the stomach *Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

1) Insulin

Insulin. Stomach

Time Rat#

(min) 1 #2 #3 #4 #5 #6 #7 #8 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.9 0.0 0.0 0.0

15 114.5 72.8 80.9 12.6 210.0 12.5 118.7 158.5 97.6 68.1 24.1 69.8%

30 95.3 19.3 35.5 12.5 211.0 12.5 15.1 66.0 58.3 68.4 24.2 117.2%

45 62.8 12.5 894.0 12.5 213.0 12.5 15.0 12.5 154.3 306.7 108.5 198.8%

60 18.2 12.5 157.0 12.5 174.0 140.3 12.5 12.5 67.4 74.7 26.4 110.9%

90 12.5 12.5 12.5 12.5 61.3 12.5 12.5 74.2 26.3 25.8 9.1 98.1%

AUCV₊₉O 4780 2132 18970 1127 14438 4001 2795 5046 6661 6451 2281 96.8% (two rat data were removed, rat #1-2 stomach)

2) Glucose

Change from base line Stomach

Time

(min) Rat# l #2 #3 #4 #5 #6 #7 #8 Mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0

15 -9.9 -21.0 -12.6 -38.1 -6.6 -24.8 3.0 -13.9 -15.5 12.5 4.4 -80.6%

30 -44.3 -43.8 -25.8 -56.1 -9.8 -54.4 -3.4 -47.6 -35.7 20.2 7.1 -56.6%

45 -75.0 -50.9 -30.2 -73.6 -17.1 -73.6 -14.8 -64.2 -49.9 25.8 9.1 -51.6%

60 -80.7 -51.3 -31.1 -66.1 -18.7 -72.8 -21.9 -55.1 -49.7 23.5 8.3 -47.3%

90 -62.3 -32.1 -35.2 -59.7 -20.3 -63.6 -29.5 -28.3 -41.4 17.5 6.1 -42.3% (two rat data were removed, rat #1-2 stomach)

Table 3

Direct dosing of coprocessed microparticles to the jejunum data (Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

1) Insulin

Insulin Jejunum

Time

(min) #9 #10 #11 #12 #13 #14 #15 #16 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0 15 413.2 1193.3 669.4 1177.5 2270.6 228 9 954.4 374.9 910.3 661.1 233.8 72.6% 30 354.3 148.4 70.5 64.7 481.0 168.4 782.9 57.4 265.9 258.2 91.3 97.1% 45 79.5 28.0 20.5 26.5 170.8 148.6 531.6 12.5 127.3 174.3 61.6 137.0% 60 23.1 14.7 12.5 16.8 71.6 117.0 200.1 12.5 58.5 68.4 24.2 116.9% 90 12.5 12.5 12.5 12.5 30.8 12.5 37.5 12.5 17.9 10.2 3.6 56.8%

AUC₀ 13506 21158 11969 19690 46003 11102 39192 7235 21232 14054 4969 66.2%

(two rat data were removed, rat# 2 jejunum and rat# 4 jejunum)

2) Glucose

Change from base lme Jejunum

Time

(min) #9 #10 #11 #12 #13 #14 #15 #16 Mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0

15 -50.8 -35.6 -16.7 -16.0 -61 .1 -38.2 -62.8 -36.3 -39.7 17.9 6.3 -45.0%

30 -67.3 -65.5 -59.5 -35.8 -81 .8 -62.5 -78.1 -52.9 -62.9 14.4 5.1 -22.9%

45 -64.4 -68.5 -76.3 -56.4 -74 .4 -71.6 -78.1 -53.3 -67.9 9.2 3.2 -13.5%

60 -62.7 -62.5 -71.3 -62.7 -62 .1 -65.6 -74.0 -42.2 -62.9 9.5 3.4 -15.1%

90 -62.4 -49.8 -69.7 -55.6 -41 .8 -44.2 -69.4 -26.7 -52.5 14.9 5.3 -28.3%

(two rat data were removed, rat# 2 jejunum and rat# 4 jejunum)

Table 4

Direct dosing of coprocessed microparticles to the stomach (Insulin (0.25mg/kg), Delivery Agent (37.5mg/kg))

D Insulin stomach

Time

(min) sto-1 sto-2 sto-3 sto-4 sto-5 sto-6 sto-7 sto-8 mean SD SEM CV

0 12.5 12.5 41.2 21.5 12.5 12.5 12.5 12.5 17.2 9.5 3.4 55.4%

15 12.5 20.8 14.7 12.5 12.5 12.5 26.4 12.5 16.0 4.9 1.7 30.7%

30 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

45 48.8 12.5 12.5 12.5 12.5 12.5 12.5 12.5 17.0 12.0 4.3 70.4%

60 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

AUCo-»₉o 1670 1250 1373 1193 1125 1125 1334 1125 1274 187 66.0 14.6&

2) Glucose

Change from base line Stomach Time Rat#

(min) 1 #2 #3 #4 #5 #6 #7 #8 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0.0

15 -2.2 -12.3 2.2 0.8 -2.2 -12.3 2.2 0.8 -2.9 5.7 2.0 196.5%

30 -14.4 -10.1 -6.5 0.8 -14.4 -10.1 -6.5 0.8 -7.6 5.6 1.9 -73.7%

45 -15.8 -8.8 -12.7 0.0 -15.8 -8.8 -12.7 0.0 -9.3 5.9 2.1 -63.4%

60 -15.8 -11.4 -17.9 -5.8 -15.8 -11.4 -17.9 -5.8 -12.7 4.6 1.6 -36.2%

90 -19.1 -16.3 -8.6 -6.6 -19.1 -16.3 -8.6 -6.6 -12.7 5.2 1.8 -40.9%

Table 5

Direct dosing of coprocessed microparticles to the jejunum data (Insulin (0.25mg/kg), Delivery Agent (37.5mg/kg)) D Insulin jejunum

Time

(min) jej-l Jej-2 Jej-3 Jej-4 jej-5 jej-S Jej-7 Jej-8 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

15 428.5 532.4 232.6 12.5 62.0 186.6 79.6 160.5 219.2 170.7 60.4 77.8%

30 67.9 100.8 44.7 12.5 15.5 12.5 14.2 49.3 39.7 30.4 10.7 76.4%

45 16.8 40.8 26.6 12.5 12.5 12.5 12.5 12.5 18.4 9.7 3.4 52.6%

60 12.5 24.7 17.3 12.5 12.5 12.5 12.5 12.5 14.6 4.1 1.5 28.1%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0%

AUCo-,₉₀ 8261 10947 5229 1125 1913 3737 2157 3897 4658 3392 1199 72.8%

2) Glucose

Change from base line Jejunum

Time

(min) #9 #10 #11 #12 #13 #14 #15 #16 Mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0.0

15 -29.7 -20.5 -35.8 1.3 -28.9 -18.2 -25.9 -25.6 -22.9 11.2 3.9 -48.9%

30 -52.2 -54.5 -41.9 -1.3 -50.0 -60.3 -36.8 -52.8 -43.7 18.7 6.5 -42.8%

45 -63.6 -65.3 -43.8 -0.7 -56.8 -69.2 -40.8 -59.7 -59.0 22.3 7.8 -37.8%

60 -56.9 -69.4 -33.8 11.6 -56.8 -62.1 -27.2 -55.7 -41.9 28.1 10.6 -67.1%

90 -63.9 -59.7 -26.9 8.3 -50.0 -31.8 -11.8 -28.4 -28.6 22.7 8.5 -79.4%

b. Results from Oral Gavage Experiments Using Tablet and Capsules

[155] The glucose and insulin data from the three formulations tested are shown in Figures 4 and 5, respectively. The individual data are listed in Tables 6 to 7. The results from the direct dosing studies to the stomach and jejunum are included for comparison. The individual glucose and insulin data for the simple mix of insulin and delivery agent is shown in Table 8.

[156] In the group of 10 rats that was dosed with capsules containing microparticles of coprocessed insulin and carrier, the average minimum glucose lowering was 70% from baseline at 30 minutes. One rat died at 15-30 minutes, likely due to hypoglycemia, six rats were rescued at 30 minutes with dextrose, an additional rat was rescued at 60 minutes, and two of the six that were rescued at 30 minutes died after 60 minutes. There were no signs of GI irritation or GI damage from the oral gavage procedure from necropsies of the rats after the experiment. The average minimum glucose lowering from tablets that contained the same amounts of insulin and carrier was 50 % .

[157] The corresponding insulin concentrations are shown in Figure 5. Insulin concentration is highest from the coprocessed microparticles in a capsule, followed by the tablet and the capsule of the simple mix. [158] In the oral gavage studies using capsules containing coprocessed microparticles, two (of 10) rats were found to exhibit high insulin absorption. Retainer samples were reassayed and insulin levels were approximately the same as those from the original samples, as shown in Table 6(3), shown above. Insulin levels with and without two high responders are shown in Figures 6 and 7, respectively. The individual and average insulin and glucose profiles from N = 10 and N=8 are shown in Figures 13 to 16.

Table 6 Oral gavage of tablets: Insulin (0.5mg/kg), Delivery Agent (75mg/kg)

1) Insulin

Time Rat#

(min) 11 #12 #13 #14 #15 #16 #17 #18 #19 #20 mean SD SEM CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.8 12.5 12.5 0.1 0.0 0.8%

15 468.8 162.1 700.1 12.5 1363.4 197.4 565.4 57.0 114.4 12.5 365.4 426.3 134.8 116.7%

30 90.5 14.5 108.6 12.5 174.0 14.4 62.7 117.5 20.0 16.1 63.1 57.2 18.1 90.7%

45 15.2 32.0 22.9 12 5 44.5 12.5 16.3 43.3 12.5 12.5 22.4 12.9 4.1 57.6%

60 12.5 12.5 13.9 12.5 23.2 16.7 12.5 12.5 12.5 12.5 14.1 3.5 1.1 24.6%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0.0 0.0 0.0% ϋCo_₉o 9180 3692 13068 1125 24532 4022 10229 3830 2768 1179 7363 7259 2295 98.6%

2) Glucose

Change from baseline

Time Rat# •

(min) 1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0 0 0.0 0.00%

15 -51.4 -38.9 -36.5 -37.8 -23.8 -26.7 12.1 16.5 25.0 54.3 -10.7 35.0 11.1 -326.7%

30 -70.8 -69.4 -66.2 -68.3 -67.9 -68.9 -57.1 -41.8 -20.7 47.8 -48 3 37.4 11.8 -77.4%

45 -66.7 -63.9 -64.9 -57.3 -57.1 -52.2 -44.0 -27.5 -27.2 52.2 -40.9 35.7 11.3 -87.4%

60 -54.2 -47,2 -41.9 -42.7 -34.5 -33.3 -9.9 -4.4 -1.1 51.1 -21 8 31.7 10.0 -145.1%

90 -50.0 -26.4 -17.6 -19.5 -13.1 5.6 9.9 11.0 32.6 106.5 3.9 42.9 13.6 1099.9%

10

Table 7

Oral gavage of capsules containing coprocessed insulin and delivery agent data (Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

5 D Insulin

Time Rat# min) 11 #12 #13 #14 #15 #16 #17 #18 #19 #20 mean SD SEM CV

0 12 5 12.5 12.5 12.5 12.5 12.5 12 5 12.5 12.5 12.5 0.0 0.0 0.0 0.0%

15 461.1 35.2 400.0 1080.6 143.1 36 7 7970 5 1611.3 369.8 922.5 1303.1 2396.7 757.9 183 9%

30 244.0 120 0 3268.4 35.3 32.7 5915.1 201.0 150.9 240.3 1134.2 2070.4 654 7 182.5%

45 26.9 13.7 3132 7 18 9 201 2 3309.5 58.7 12.5 38.3 756.9 1399.0 442.4 184.8%

60 13.8 12.5 2129.7 12.5 28.7 3693.9 16.0 12.5 12.5 659 1 1335 7 422.4 202.6%

90 12.5 12 5 984.9 12.5 12.5 12.5 12.5 151.4 367.5 116.2 242.7%

[n=9) 11752 3096 175011 3522 4986 285725 28279 8561 18579 59946 100827 33609 168.2%

(n=7) 11752 3096 3522 4986 28279 8561 18579 11254 9279 3507 82.4%

2) Glucose

Time

(mm) #11 #12 #13 #14 #15 #16 #17 #18 #19 #20 mean. SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0 0 0.0 0 0%

15 -27 9 41.6 -32.5 -30.4 -23 3 19.8 -53.9 -5.5 -19.6 -25 3 -15.7 27. .7 9.8 -176 .3%

30 -79 0 -17 5 -76.6 -64 0 -40.1 -78.0 -77.0 -63.8 -77 0 -63.7 21. ,4 7.5 -33 .5%

45 -42 1 -464 -62.6 -36 7 -66.7 -53.9 -4.9 10.9 -50.0 -39.2 25 9 9.1 -66 2%

60 3.2 -34.9 -61.4 -13 3 -75 3 -70.3 -7.2 64.5 -48 3 -30.8 45. .9 16.2 -149 0%

90 68 4 -7 2 -62.6 20 7 92.0 105.1 -8.6 29.3 70. .0 24.7 239 0%

3) Reassay insulin levels of Rats 14 and 17

10 Rat #14

Rat #17

Table 8

Oral gavage of capsules containing containing a simple mix of insulin and delivery agent data (Insulin (0.5mg/kg), Delivery Agent (75mg/kg))

1) Insulin rat time #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean SD SE CV

0 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

15 41.4 230.9 58.3 110.7 82.2 12.5 12.5 12.5 14.9 25.2 60.1 68.8 21.8 114.4%

30 12.5 49.6 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 16.2 11.7 3.7 72.3%

45 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

60 12.5 . 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

90 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 12.5 0 0 0.0%

AUC₀, ->Qn 1559 4958 1812 2598 2171 1125 1125 1125 1161 1316 1895 1189 376 62.7%

2) Glucose Change from base line

Time RaW

(min) 1 #2 #3 #4 #5 #6 #7 #8 #9 #10 mean SD SEM CV

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0%

15 7.7 -32.3 3.4 9.3 14.5 58.7 34 1.7 51.7 1.7 15.0 26.7 8.4 177.7%

30 -38.2 -78.5 -46.1 -42.9 -47.2 28.7 45.7 54.6 2.7 -17.9 -13.9 44.9 14.1 -322.4%

45 -13.9 -22.6 -20.8 -34.8 -38.9 20.4 42.1 44.8 -0.7 11.0 -1.3 30.1 9.5 -2247.0%

60 -3.1 41.4 -18.5 -14.3 -26.9 20.9 65.5 47.7 26.2 32.9 17.2 31.3 9.9 182.1%

90 6.2 73.1 32.6 53.4 40.1 43.1 51.7 39.6 30.6 41.2 18.4 5.8 44.6%

Example 2

1. Summary of Intravenous, Portal Vein and Subcutaneous Experiments a. Experiment [159] Intravenous, intraportal and subcutaneous dosing in rodents were conducted to estimate the absolute bioavailability, the absorption of insulin in the portal vein, and the bioavailability to relative subcutaneous administration. The data are summarized in Tables 9 to 11. The average insulin AUCo- WDose was 0.0093 min.kg/ml from intravenous dosing. This value was assumed to be constant in the estimates of absolute bioavailability. Table 9 Insulin pharmacokinetics results from intravenous (IV) administration

AUCo-*»/ AUCo^₀/

Dose C₀ K₁ AUC_0-^ Dose C₀->_∞/ Dose Dose (min.

(μg/kg) (ng/ml) (min ¹) ng/ml) (min. kg/ml) (ml/kg) (kg/ml) (min. kg/ml)

2.5 15.79 0.89 17.74 0.0071 158 0.0063 0.0071 2.5 7.48 0.71 10.56 0.0042 334 0.0030 0.0042 2.5 13.36 0.84 15.87 0.0063 187 0.0053 0.0063 2.5 12.78 0.72 17.73 0.0071 196 0.0051 0.0071

2.5 11.26 0.72 15.75 0.0063 222 0.0045 0.0063

XtSD 12.1 ± 0.78 ± 0.0062 ± 219 ± (n=5) 2.8 0.07 15.5 ± 2.6 0.0011 68

50.67 0.51 99.35 0.0124 157.88 0.0063 0.0124 52.64 0.53 99.32 0.0124 151.98 0.0066 0.0124

8 49.63 0.48 103.4 0.0129 161.19 0.0062 0.0129

XiSD 51.0 ± 0.51 ± 100.7 ± 0.0126 ±

(n=3) 1.5 0.03 2.4 0.0003 157 ± 5

46.79 0.47 99,55 0.0111 192.35 0.0052 0.0111 36.32 0.48 75.67 0.0084 247.80 0.0040 0.0084

9 55.30 0.45 122.89 0.0137 162.75 0.0061 0.0137

X±SD 46.1 ± 0.47± 99.4 ± 0.0110 ± 201 ±

(n=3) P.5 0.02 23.6 0.0026 43

Average 0.0053 0.0093

SD 0.001133858 0.0033

SEM 0.000341871 0.00099

Table 10 Insulin results from portal and systemic administrations

Table 11 Insulin results from subcutaneous (SC) administration dy Insulin AUC₀- AU(W

# dose Time Insulin SD SE Glucose SD SE N >τ -* max ^ max AUC₀^_x Dose

(min. (min. (min.

(mg/kg) (uU/ml) (mg/dl) uU/ml) (min) ng/ml) kg/ml)

1 0.025 0 0.49 0.77 0.31 121 12.6 5.1 6 3640.8 15 76.1 140.0 0.005601

15 76.14 17.52 7.15 110 26.4 10.7 6

30 54.34 21.92 8.95 90 31.4 12.8 6

45 32.26 4.59 1.87 101 36.6 14.9 6

60 21.61 4.29 1.75 102 30.4 12.4 6

120 5.61 2.93 1.19 130 37.0 15.1 6

180 1.64 1.82 0.74 194 54.7 22.3 6

2 0.025 0 2.15 2.37 0.96 116 12.9 5.2 6 4234.4 15 79.5 162.9 0.006514

15 79.54 24.22 9.88 103 8.3 3.3 6

30 53.03 20.27 8.27 90 17.4 7.1 6

45 45.53 23.98 9.79 100 26.2 10.7 6

60 23.48 21.47 8.76 120 23.6 9.6 6

120 9.36 9.60 3.92 196 45.6 18.6 6

180 3.48 5.23 2.13 188 45.1 18.4 6

3 0.05 0 1.51 1.59 0.71 83 6.9 3.1 5 7230.0 15 98.8 278.1 0.005562

15 98.85 14.16 6.33 66 17.1 7.6 5

30 73.59 25.49 11.40 60 21.4 9.5 5

45 61.37 21.56 9.64 61 27.2 12.1 5

60 58.20 32.09 14.35 73 24.4 10.9 5

120 21.34 17.43 7.79 98 21.6 9.7 5

180 8.30 7.49 3.35 105 30.7 13.7 5

4 0.05 0 0.37 0.90 0.36 73 4.8 1.9 6 6407.7 15 103.4 246.5 0.004929

15 103.42 25.17 10.27 66 6.9 2.8 6

30 81.64 24.94 10.18 59 10.5 4.3 6

45 68.91 20.41 8.33 64 9.0 3.6 6

60 58.58 21.87 8.93 72 7.0 2.8 6

90 32.01 9.92 4.05 88 14.0 5.7 6

120 21.15 9.42 3.84 110 17.0 6.9 6 0.05 0 0 0 0 60 5.1 2.2 5 1317.0 15 34.9 50.7 0.001013

15 34.90 46.01 20.57 42 6.1 2.7 5

30 9.71 18.15 8.12 38 6.9 3.1 5

45 16.57 37.04 16.56 42 9.8 4.4 5

60 10.46 20.05 8.96 48 17.6 78 5

90 2.94 4.25 1.90 52 25.2 11.3 5

120 5.05 6.91 3.09 66 41.8 18.7 5

0.05 0 12.80 18.01 7.35 68 5.0 2.0 6 9937.5 15 191.7 382.2 0.007644

15 191.67 100.40 40.98 40 6.2 2.5 6

30 119.71 79.53 35.56 39 2.5 1.1 5

45 121.33 118.89 48.53 36 7.1 2.9 6

60 74.65 57.97 23.66 32 4.3 1.7 6

90 42.88 37.77 15.42 35 5.5 2.2 6

120 25.65 17.59 7.18 39 9.2 3.7 6

0.05 0 5.85 3.11 1.27 65 8.7 3.5 6 7711.1 30 126.0 296.6 0.005932

15 90.29 73.70 30.08 45 7.2 2.9 6

30 125.96 107.54 43.90 39 8.9 3.6 6

45 67.83 95.55 39.01 40 10.9 4.4 6

60 78.19 105.69 43.14 42 13.2 5.4 6

90 45.77 49.52 20.22 42 18.7 7.6 6

120 18.24 13.53 5.52 50 26.2 10.7 6

0.05 0 2.40 5.88 2.40 70 4.8 1.9 6 5303.3 15 131.7 204.0 0.004079

15 131.71 131.15 53^'54 59 6.8 3.0 6

30 70.90 45.95 18.76 64 12.1 4.9 6

45 66.28 42.41 17.31 69 16.5 6.7 6

60 0.59 1.27 0.52 73 13.1 5.3 6

90 33.96 14.99 6.12 89 17.8 7.2 6

120 14.64 9.35 3.82 106 18.6 7.6 6

Avg

AUC₀->τ/

Dose 0.005159

(nun. kg/ml)

b. Results

[160] The ratio of systemic to portal insulin was found to be approximately 0.62 (calculated from data in Table 10). Hence, the bioavailability in the portal vein can be calculated by dividing the absolute bioavailability by 0.62. The portal bioavailability provides an estimate of drug absorption from oral delivery. The average insulin AUCo- _>t/Dose was 0.00516 min.kg/ml from subcutaneous dosing. This value is used to estimate bioavailability relative to subcutaneous. With the exception of the intravenous data, all AUC were calculated from t=0 to the last sampling point (i.e. AUCo->t). [161] In the rat model, these results from intraportal administration suggest that the maximum absolute bioavailability of insulin is approximately 60% from oral delivery or by any other means of 100% GI absorption of insulin into the portal vein. Secondly, the absolute bioavailability from SC is approximately 56%. [162] The estimates of bioavailability (absolute bioavailability, portal bioavailability, relative bioavailability to subcutaneous, and relative portal bioavailability to subcutaneous) are summarized in Figures 8 to 12, and in Table 12. The estimated absolute bioavailability from in situ dosing to the stomach and the jejunum are shown in Figure 8. The values of bioavailability were 5 % when dosed in the stomach and 18 % when dosed in the jejunum from microparticles containing coprocessed insulin (0.5 mg/kg) and delivery agent (75 mg/kg).

[163] The estimated absolute bioavailability from the tablet and capsule formulations dosed by oral gavage in rats are shown in Figure 9 using formulations containing 0.5 mg/kg insulin and 75 mg/kg delivery agent. The values of bioavailability were 6% when dosed from tablets, and 1.6% when dosed from capsules containing a simple mix of insulin and carrier.

Table 12 Estimates of bioavailability

SC 0.0052 55.67

Insulin (0.5mg/kg) + Delivery Agent (75mg/kg) Stomach

4778.70 15 114.50 183.80 3.68E-04 3.97 6.₄0 7.12 11.49 213075 15 7275 81 95 1 64E-04 1 77 2 85 3 18 5 12

1895858 45 89386 729 18 1 46E-03 1574 25 38 2827 45 59

112665 15 1261 43 33 867E 05 094 1 51 1 68 2 71

14425 95 45 21273 554 84 1 11E-03 11 97 19 31 21 51 3469

400073 60 14031 153 87 3 08E-04 332 5 36 5 96 962

279345 15 11866 10744 2 15E 04 232 374 4 16 672

504623 15 15850 194 09 3 88E 04 4 19 676 7 52 12 13

Mean 665763 28 13 21549 25606 5 12E 04 553 891 993 1601

SD 6446 07 18 70 28036 247 93 4 96E-04 5 35 863 9 61 15 50

SE 2279 03 6 61 99 12 87 65 1 75E-04 1 89 3 05 3 40 5 48

CV

(%) 96 82 66 48 130 10 9682 9 68E+01 96 82 96 82 96 82 96 82

Jejunurr 1

13504 88 15 41323 51942 1 04E-03 11 21 18 08 20 14 3248

21156 68 15 119325 813 72 1 63E-03 17 56 28 32 31 54 5088

11967 90 15 66938 460 30 9 21E-04 9 93 16 02 17 84 2878

19689 45 15 117747 75729 1 51E-03 16 34 26 36 2936 4735

46001 85 15 2270 55 1769 30 3 54E-03 38 18 61 59 68 59 11062

11101 35 15 228 90 42698 854E-04 9 21 1486 16 55 2670

39190 05 15 95435 1507 31 301E-03 32 53 5247 58 43 9424

723405 15 374 88 278 23 556E-04 600 968 10 79 1740

Mean 21230 78 15 910 25 81657 1 63E-03 1762 2842 31 65 51 05

SD 14053 61 0 661 14 54052 1 08E-03 11 66 18 81 20 95 33 80

SE 4968 70 0 233 75 191 10 3 82E-04 4 12 665 741 11 95 c L_*~\ Vr

(%> 66 19 0 72 63 66 19 6 62E+01 66 19 66 19 66 19 66 19

Tablet

9181 20 15 468 81 353 12 706E-04 762 12 29 13 69 22 08

3692 64 15 162 11 14202 2 84E-04 307 494 5 51 888

1306889 15 700 10 50265 1 01E-03 1085 1750 1948 31 43

112500 0 12 50 4327 865E-05 093 1 51 1 68 2 71

24533 57 15 1363 43 94360 1 89E-03 2036 3284 36 58 59 00

402233 15 19739 15470 309E-04 334 538 600 967

1022804 15 565 39 393 39 7 87E-04 849 1369 1525 2460

383006 30 11747 14731 2 95E-04 3 18 5 13 5 71 921

276770 15 11435 10645 2 13E 04 230 371 4 13 666

117843 30 1606 4532 9 06E-05 098 1 58 1 76 2 83

Mean 736278 1650 371 76 283 18 566E-04 611 986 1098 1771

SD 766989 901 44561 29500 5 90E-04 637 1027 11 44 18 44

SE 242543 285 14091 9329 1 87E 04 201 325 3 62 5 83

(%> 10417 5463 11986 104 17 1 04E+02 10417 10417 104 17 104 17

Capsule (co-dried)

(N=9) 1157064 15 461 06 445 02 8 9OE 04 960 1549 17 25 27 82

309624 30 12002 11909 2 38E 04 257 4 15 462 745

175011 18 30 326840 6731 20 1 35E-02 14527 23430 26093 420 86 352346 15 14314 13552 271E-04 292 472 525 847

498801 45 20124 19185 384E-04 414 668 744 1199

26572526 15 797050 1098943 22OE 02 23716 38252 42600 68710

2827849 15 161131 108763 218E-03 2347 3786 4216 6800

855993 15 38978 32923 658E 04 711 1146 1276 2058

1857854 15 92250 71456 143E-03 1542 2487 2770 4468

Mean 5992575 2167 167422 230484 000 4974 8023 8935 14411

SD 10083827 1090 257042 387839 001 8370 13500 15034 24249

SE 3361276 363 85681 129280 000 2790 4500 5011 8083

CV

(%) 16827 5029 15353 16827 16827 16827 16827 16827 16827

Capsule (co-dried)

(N=7) 1157064 15 46106 44502 89OE 04 960 1549 1725 2782

309624 30 12002 11909 238E 04 257 415 462 745

352346 15 14314 13552 271E04 292 472 525 847

498801 45 20124 19185 384E 04 414 668 744 1199

2827849 15 161131 108763 218E 03 2347 3786 4216 6800

855993 15 36978 32923 658E 04 711 1146 1276 2058

1857854 15 92250 71456 143E 03 1542 2487 2770 4468

Mean 1122790 2143 54701 43184 000 932 1503 1674 2700

SD 927729 1180 54429 35682 000 770 1242 1383 2231

SE 350649 446 20572 13486 000 291 469 523 843

CV

(%) 8263 5508 9950 8263 8263 8263 8263 8263 8263

Capsule (simple mix)

155808 15 4137 5993 12OE 04 129 209 232 375

495666 15 23089 19064 381E04 411 664 739 1192

181260 15 5834 6972 139E-04 150 243 270 436

259835 15 11072 9994 2 OOE-04 216 348 387 625

217106 15 8224 8350 167E-04 180 291 324 522

112500 0 1250 4327 865E-05 093 151 168 271

112500 0 1250 4327 865E-05 093 151 168 271

112500 0 1250 4327 865E-05 093 151 168 271

116165 15 1494 4468 894E-05 096 156 173 279

131597 15 2523 5061 101E-04 109 176 196 316

Mean 189494 1050 6012 7288 146E-04 157 254 283 456

SD 118869 725 6882 4572 914E-05 099 159 177 286

SE 37590 229 2176 1446 289E-05 031 050 056 090

CV

(%) 6273 6901 11447 6273 627E+01 6273 6273 6273 6273

Insulin (0 25mg/kg) *^■ Delivery Agent (37.5mg/kg) Stomach

16695D 45 4880 6421 257E-04 277 447 498 803

124950 15 2080 4806 192E-04 207 335 373 601

137325 0 4120 5282 211E04 228 368 409 660

119250 0 2150 4587 183E 04 198 319 356 574 112500 0 1250 4327 173E-04 187 301 335 541

112500 0 1250 4327 173E-04 187 301 335 541

133322 15 2638 5128 205E-04 221 357 398 641

112500 0 1250 4327 173E-04 187 301 335 541

Mean 127412 938 2452 4900 196E-04 212 341 380 613

SD 18656 1591 1377 718 287E 05 031 050 056 090

SE 6596 563 487 254 101E 05 011 018 020 032 CV

(%) 1464 16971 5616 1464 146E+01 1464 1464 1464 1464

Jejunum

826050 15 42850 31771 127E-03 1371 2212 2463 3973

1094700 15 53240 42104 168E-03 1817 2931 3264 5265

522900 15 23260 20112 804E-04 868 1400 1559 2515

112500 0 1250 4327 173E-04 187 301 335 541

191094 15 6195 7350 294E-04 317 512 570 919

373593 15 18656 14369 575E-04 620 1000 1114 1797

215720 15 7960 8297 332E-04 358 578 643 1038

389703 15 16054 14989 600E-04 647 1043 1162 1874

Mean 465732 1313 21183 17915 717E-04 773 1247 1389 2240

SD 339258 530 18248 13048 522E-04 563 908 1012 1632

SE 119946 188 6452 4613 185E-04 199 321 358 577 CV (%) 7284 4041 8614 7284 728E+01 7284 7284 7284 7284

Example 3 Insulin and 4-CNAB Stability in Simulated Gastric Fluid [164] The stability of insulin in simulated gastric fluid (SGF) was evaluated in the presence and absence of 4-CNAB. Solutions were prepared containing insulin (1 mg/ml) with and without monosodium 4-CNAB (1 mg/ml).

[165] The SGF was prepared with and without pepsm, a gastric enzyme. SGF pH 1.2 was prepared as per the USP NF 26 guidelines. 2 g sodium chloride and 3.2 g of pepsin were weighed and added to a suitable container, and deionized water was added to reach one liter in volume. If necessary, the pH was adjusted to 1.2 by addition of concentrated HCl or NaOH. A second SGF solution omitting the pepsm was also prepared. [166] Four 50 ml samples of SGF (two with pepsin and two without) were placed into a jacketed vessel connected to a circulating water bath set at 37°C. The solutions were stirred with magnetic stir bars for ten minutes to allow the solutions to reach 37⁰C and reach thermal equilibrium. 50 mg of 4-CNAB was added to one of the samples containing pepsin and one of the samples without pepsin, and the solutions were stirred for a few minutes to allow the 4-CNAB to dissolve. 50 mg of insulin was added to the each of the samples. After dissolution of the insulin, samples of the solutions were taken at predetermined time intervals, filtered, and immediately assayed by HPLC for insulin and 4- CNAB content. The first sample withdrawn after all the insulin was dissolved was considered to have been drawn at time zero (0). The results are shown in table 13.

Table 13

[167] The term " % of theoretical" as used herein, means the percent of the concentration (mg/mL) of withdrawn solution at the time-point the sample was taken as compared to the theoretical concentration (mg/mL) of the measuring component for experiment. The standard of deviation for the HPLC analysis is +5 % . These results show that insulin is unstable in SGF containing pepsin, since only 3.0% of the insulin remained at the first sampling point (97% of the insulin was degraded), while insulin is stable at least up to 2 hours in SGF without pepsin.

Example 4 Stability of Insulin in Simulated Intestinal Fluid

[168] The stability of insulin in simulated intestinal fluid (SIF) was evaluated in the presence and absence of 4-CNAB.

[169] The SIF solutions were prepared with and without pancreatic enzyme. SIF pH 7.5 was prepared as per the USP NF 26 guidelines. SIF was prepared by addition of 6:8 g monobasic potassium phosphate and 10 g of pancreatin into a suitable vessel, and deionized water was added to reach a total volume of one liter. If necessary, the pH was adjusted to 7.5 by addition of 0.2 N sodium hydroxide. A second SIF solution omitting the pancreatin, an intestinal enzyme, was also prepared.

[170] Four 50 ml samples of SIF (two with pancreatin and two without) were placed into a jacketed vessel connected to a circulating water bath set at 37⁰C. The solutions were stirred with magnetic stir bars for ten minutes to allow the solutions to reach 37⁰C and reach thermal equilibrium. 50 mg of 4-CNAB was added to one of the samples containing pepsin and one of the samples without pepsin, and the solutions were stirred for a few minutes to allow the 4-CNAB to dissolve. 50 mg of insulin was added to the each of the samples. After dissolution of the insulin, samples of the solutions were taken at predetermined time intervals, and immediately assayed by HPLC for insulin and 4-CNAB content. The results are shown in table 14.

Table 14

[171] These results show that insulin is stable in SIF without pancreatin and degrades in presence of the enzyme. Insulin is more stable in SIF with and without enzyme than in SGF with and without enzyme. At the first sampling time point (0 minuts) only 3.0% insulin remained in SGF with enzymes while 58.9% and 66.9% insulin remained in SIF.

Example 5 Effect of Formulation on Insulin Absorption and Action

[172] Six formulations containing insulin shown in Table 15 were prepared as follows.

Table 15

[173] Polyplasdone XL, is available from International Specialty Products, Wilmington DE.; Emcocel HD90, Prosolv HD90, Emcompress and Anhydrous Emcompress is available from JRS Pharma, Patterson, NY. [174] The formulations were fed to rhesus monkeys in doses containing 100 mg/kg of 4-CNAB and 13 U/kg insulin. Groups of four rhesus monkeys, two males and two females, were fasted for at least 12 hrs prior to dosing and up to 4 hrs after dosing. Water was withheld approximately 1 hr before dosing and up to 2 hrs after dosing after which it was permitted ad libitum. The dosing was followed by a 5 ml water flush. Blood samples (approximately 2 ml each) were collected by venipuncture at 15 minutes before dosing and at 5, 10, 15, 20, 30, 45 minutes and 1, 1.5, 2, 3, 4 hr after dosing. Each blood sample was divided into two portions. One portion was allowed to clot at room temperature and centrifuged at 2-8°C for 10 minutes at 3000 rpm. The serum obtained was aliquoted into two portions and stored at -70°C until shipment. One sample was shipped to Emisphere on dry ice for insulin analysis by ELISA while the other was retained by the CRO for serum glucose analysis. The second portion of the blood was kept on wet ice for up to 30 minutes and centrifuged at 2-8°C for 10 minutes at 3000 rpm. The plasma obtained was shipped to Emisphere on dry ice for analysis of 4-CNAB content by HPLC. Each formulation was administered to 4 rhesus monkeys, except formulation 1, which was administered to 8 rhesus monkeys. Blood samples were taken at predetermined intervals as described above and assayed for insulin and glucose levels. The results are shown in table 16 and in Figures 17 and 18 . Table 16

[175] Disintegration time was determined in water at 37 ± 2^DC using the method described in USP <701>. Multiple tubes containing water are placed in a basket-rack assembly immersed in a water bath maintained at 37 ± 2°C. The basket-rack assembly raises and lowers the tubes at a constant frequency. The tablets are placed in the tubes and are periodically examined to determine if they have disintegrated completely. Each tablet is tested in six different tubes. If 1 or 2 tablets fails to consistently disintegrate, the procedure is repeated on additional tablets. The average maximum concentration of insulin (C_m3x) was determined for each group based upon the serum levels of insulin measured as described above. If the blood glucose levels in the primates falls to very low levels (< 1 mmol/L) during the experiment they are administered dextrose in order to bring the blood glucose up to a safe level. The average Cmax for each group, as well as the number of rhesus monkeys rescued, is shown in table 17.

Table 17

Example 6 Preparation of Enteric Coated Tablets

[176] Capsules were manufactured by encapsulating 300 mg of a formulation including 150 units insulin, 200 mg 4-CNAB, 0.4% w/w povidone, —29.1% w/w Emcompress, 1% w/w SLS, and 1% w/w magnesium stearate into size 2 white opaque capsules. The capsules were first coated with a subcoat consisting of Opadry clear for a weight gain of 5% followed by an enteric coat of 20% weight gain for a total weight gain on the capsules of 25 % .

[177] Tablets were manufactured by pressing 300 mg of the formulation described above into tablets. An 10% weight gain enteric coat was applied. The formulations for the subcoats and enteric coats are shown in table 18 below.

Table 18

[178] Opadry™ Clear is available from Colorcon, of West Point, PA.

[179] Milli Q Water is highly purified water and is available from Millipore of Billerica, Massachusetts.

[180] Eudragit L30D55 is available from Degussa AG, Parsippany, NJ.

[181] To verify the effectiveness of the enteric coat, the coated capsules and tablets were placed in 0.1 N HCl for two hours or pH 6.8 phosphate buffer for one hour. The coated capsules and tablets did not dissolve in the 0.1 N HCl, but did dissolve in the pH 6.8 phosphate buffer.

Example 7 SNAC Micro Beads Coated With Heparin

[182] 5 g of SNAC and 0.5 g of magnesium stearate were mixed. 0.02 g of the mixed powder was fed into a die. Small beads of SNAC and magnesium stearate were made at 1200 PSI bar pressure The beads had a round/ball shape size of about 0.2 mm to about 2.0 mm. The SNAC beads were then coated with 2.5 g of heparin, in liquid form, by a rotary method and dried under vacuum oven at 40° C for 10 hours.

Example 8 Micronized SNAP with Heparin

[183] SNAD was screened through a 35 mesh Tyler standard sieve. The SNAD was milled with a Glen Mills, Model SlOO centrifugal ball mill (Clifton, NJ) equipped with a 250 mL stainless steel grinding jar and 30 mm (440c) diameter stainless steel balls was used. The process parameters investigated were (1) number of balls used, (2) duration of milling, (3) milling speed, and (4) milling jar total charge. A Malvern Mastersizer 2000 equipped with a Scirocco 2000 dry accessory was used for particle size determination. A Kratos XRD 6000 (version 4.1) X-ray powder diffractometer scanning over the 2Θ range 5- 40° 2Θ was used for monitoring crystallinity changes. The diverging, scattering, and receiving slits were 1°, 1°, and 0.3 mm respectively. A Brinkmann 737 KF coulometer was used for moisture content determination while a Quantachrome Nova 3000 Series Surface Area Analyzer was used for specific surface area determination.

[184] The results indicated that the particle size distribution of pre-screened SNAD was d(0.1) = 1.6 μm, d(0.5) = 10.5 μm, and d(0.9) = 314.9 μm. The data obtained using different numbers of balls ranging from 1 to 5 indicated that the optimum number of balls for the charge used was 2. The use of 2 balls yielded the particle size d(0.1) = 1.1 μm, d(0.5) = 12.0 μm, and d(0.9) = 154.3 μm. [185] An evaluation of the effect of milling time for a fixed number of balls and charge indicated that a milling time of 120 minutes was optimum resulting in the particle size distribution, d(0.1) = 2.0 μm, d(0.5) = 15.4, and d(0.9) = 62.9 μm.

[186] An evaluation of the milling speeds 100, 300, and 500 rpm indicated that optimum milling was obtained at 300 rpm. This speed yielded the particle size distribution, d(0.9) = 62.9 μm compared to unmilled SNAD d(0.9) = 314.9 μm.

[187] A charge of 37 niL of the 250 mL milling jar provided better milling compared to 75 and 112 mL. The powder X-ray diffraction analysis indicated that milling did not result in crystallinity changes for SNAD. The Karl Fischer moisture content determination indicated no significant changes in moisture content.

[188] The SNAD was then mixed with heparin.

Example 9 Micronized SNAC with Micronized Heparin . [189] SNAC and heparin were micronized separately by the procedure described in Example 8 with 2 balls at 200 rpm for 120 minutes and then mixed together. The micronized SNAC had a d(0.5) of 7.574 μm SNAC/heparin capsules having the formulations shown in table 19 below were prepared by hand packing them into hard gelatin capsules.

Table 19

[190] ¹ - Propylene glycol monocaprylate is available as Capmul™ PG 8 from Abitec Corporation of Columbus, OH.

[191] ² - PEG 300 is available as Carbowax™ 300 from Dow Chemical Co. of Midland, MI.

[192] The heparin, SNAC, and sodium lauryl sulfate were mixed. Separately, the PEG 300, propylene glycol monocaprylate, and water (for formulation B) were mixed. 50% of the liquid PEG 300/propylene glycol monocaprylate mixture was transferred to a mortar. The heparin, SNAC₇ and sodium lauryl sulfate blended powder was added little by little and triturated with the liquid in the mortar and pestle. The capsules were then packed with the resulting mixture.

Example 10 Micronized SNAC/Heparin [193] Heparin (118.5 mg/dose (22,500 rpm)) and SNAC (125 mg/dose) were dry mixed, screened through a 35 mesh screen, and milled for about 4 minutes with a ball mill. The mixture was packed into capsules (Capsugel Size 1 capsules (Greenwood, SC)).

[194] The capsules were administered to rhesus monkeys (2 capsules per monkey) by the following procedure. Rhesus monkeys weighing between 3.5 - 5.0 kg were fasted overnight before the experiments and food was returned about 2 hours after dosing. Water was withheld from 30 minutes prior to dosing until 30 minutes after dosing, except for those quantities used for dosing. Each dosage form was delivered to the rear of the mouth using a pill gun. After release of the dosage form, 5 ml of reverse osmosis water was administered into the oral cavity to facilitate swallowing. Following delivery, the oral cavity was inspected to ensure that the capsule was swallowed. Antifactor Xa from blood samples was measured over 6 hours.

[195] The results are shown in Figure 19.

Example 11

Micronized SNAC/Heparin

[196] Capsules containing micronized SNAC/heparin as shown in table 20 below were prepared as follows.

[197] A solution of heparin and SNAC was prepared as follows. The required amounts of heparin and SNAC were weighed out and water, which was previously adjusted to a pH of about 8 with sodium hydroxide, was added. The pH of the resulting solution was in the range of about 7.3 - 7.5. The solution pH was adjusted to a pH of about 8 with sodium hydroxide. The solution was then dried in a RotoVap apparatus at 50° C under vacuum. The evaporating was done using the program outlined below.

[198] 1. Immediate reduction of vacuum from 760 torr to 200 torr [199] 2. Reduction of vacuum pressure from 200 to 100 torr in 2 minutes [200] 3. Reduction of vacuum pressure from 100 to 50 torr in 2 minutes [201] 4. Reduction of vacuum pressure from 50 to 25 torr in 4 minutes [202] 5. Reduction of vacuum pressure from 25 to 15 torr in 4 minutes [203] 6. Reduction of vacuum pressure from 15 to 10 torr in 2 minutes [204] 7. Evaporating at 10 ± 2 torr and 70 rpm in 30 minutes [205] 8. Switch to 50 rpm manually and continue with evaporating for 4 hours [206] The sample was vacuum dried overnight. The resulting powder was then micronized and filled into capsules to give the desired dose.

Table 20

[207] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[208] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

What is Claimed is:

1. Particles comprising a delivery agent and an active agent, wherein the particles have a median particle size of less than about 999 micrometers.

2. The particles of claim 1, wherein the particles have a median particle size of about 1 nanometer to about 999 micrometers.

3. The particles of claim 2, wherein the particles have a median particle size of about 1 to about 999 micrometers.

4. The particles of claim 2, wherein the particles have a median particle size of about 1 to about 999 nanometers.

5. The particles of claim 2, wherein the particles have a median particle size of about 45 to about 850 micrometers.

6. The particles of claim 2, wherein the particles have a median particle size of about 45 to about 150 micrometers.

7. The particles of claim 2, wherein the particles have a median particle size of about 150 to about 250 micrometers.

8. The particles of claim 2, wherein the particles have a median particle size of about 250 to about 425 micrometers.

9. The particles of claim 2, wherein the particles have a median particle size of about 425 to about 850 micrometers.

10. The particles of claim 2, wherein the particles have a median particle size of about 100 to about 1000 nanometers.

11. The particles of claim 10, wherein the particles have a median particle size of about 500 to about 1000 nanometers.

12. A pharmaceutical formulation comprising particles having a median particle size of less than about 999 micrometers, the particles comprising a delivery agent and an active agent.

13. The pharmaceutical formulation of claim 12, wherein the particles have a median particle size of about 1 nanometer to about 999 micrometers.

14. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 1 to about 999 micrometers.

15. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 1 to about 999 nanometers.

16. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 45 to about 850 micrometers.

17. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 45 to about 150 micrometers.

18. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 150 to about 250 micrometers.

19. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 250 to about 425 micrometers.

20. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 425 to about 850 micrometers.

21. The pharmaceutical formulation of claim 13, wherein the particles have a median particle size of about 100 to about 1000 nanometers.

22. The pharmaceutical formulation of claim 21, wherein the particles have a median particle size of about 500 to about 1000 nanometers.

23. A pharmaceutical formulation comprising a delivery agent and an active agent, wherein the delivery agent is in the form of particles having a median particle size of less than about 999 micrometers.

24. The pharmaceutical formulation of claim 23, wherein the delivery agent particles have a median particle size of about 1 nanometer to about 999 micrometers.

25. The pharmaceutical formulation of claim 23, wherein the active agent is in the form of particles, and the active agent particles have a median particle size of about 1 nanometer to about 999 micrometers.

26. The pharmaceutical formulation of claim 24, wherein the delivery agent particles have a median particle size of about 1 to about 999 micrometers.

27. The pharmaceutical formulation of claim 24, wherein the delivery agent particles have a median particle size of about 1 to about 999 nanometers.

28. The pharmaceutical formulation of claim 26, wherein the delivery agent particles have a median particle size of about 7 to about 16 micrometers.

29. The pharmaceutical formulation of claim 23, wherein the delivery agent particles are compressed to form micro-beads.

30. The pharmaceutical formulation of claim 29, wherein the micro-beads are coated with an active agent.

31. The pharmaceutical formulation of claim 30, wherein the active agent is insulin or heparin.

32. The pharmaceutical formulation of any of claims 29-31, wherein the micro- beads have a diameter of about 0.2 mm to about 2.0 mm.

33. The pharmaceutical formulation of claim 25, wherein the delivery agent particles and the active agent particles both have a median particle size of about 1 to about 999 micrometers.

34. The pharmaceutical formulation of claim 25, wherein the delivery agent particles and the active agent particles both have a median particle size of about 1 to about 999 nanometers.

35. A pharmaceutical formulation comprising a delivery agent and an active agent, wherein the active agent is in the form of particles having a median particle size of less than about 999 micrometers.

36. The pharmaceutical formulation of claim 35, wherein the active agent particles have a median particle size of about 1 nanometer to about 999 micrometers.

37. The pharmaceutical formulation of claim 35, wherein the active agent particles have a median particle size of about 1 to about 999 micrometers.

38. The pharmaceutical formulation of claim 17, wherein the active agent particles have a median particle size of about 1 to about 999 nanometers.

39. The pharmaceutical formulation of any of any of claims 12-38, wherein the delivery agent compound is selected from N-(8-[2-hydroxybenzoyl]-amino)caprylic acid, N- (10-[2-hydroxybenzoyl]-amino)decanoic acid, 8-(2-hydroxy-4- methoxybenzoylamino)octanoic acid, 8-(2-hydroxy-5-chlorobenzoylamino)-octanoic acid, 4- [(2-hydroxy-4-chlorobenzoyl)-amino]butanoic acid, and pharmaceutically acceptable salts thereof.

40. The pharmaceutical formulation of any of claims 12-38, wherein the delivery agent compound is N-(8-[2-hydroxybenzoyl]-ammo)caprylic acid or a pharmaceutically acceptable salt thereof.

41. The pharmaceutical formulation of any of claims 12-38, wherein the delivery agent compound is N-(10-[2-hydroxybenzoyl]-amino)decanoic acid or a pharmaceutically acceptable salt thereof.

42. The pharmaceutical formulation of any of claims 12-38, wherein the delivery agent compound is 4-[(2-hydroxy-4-chloro-benzoyl)-amino]butanoic acid or a pharmaceutically acceptable salt thereof.

43. The pharmaceutical formulation of any of any of claims 12-38, wherein the active agent is selected from proteins, polypeptides, peptides, hormones, and polysaccharides.

44. The pharmaceutical formulation of any of any of claims 12-38, wherein the active agent is selected from the following, including synthetic, natural or recombinant sources thereof: growth hormones; growth hormone releasing hormones; growth hormone releasing factor, interferons; interleukin-1; interleukin-2; insulin, optionally having counter ions including zinc, sodium, calcium and ammonium; insulin-like growth factor; heparin; calcitonin; erythropoietin; atrial naturetic factor; antigens; monoclonal antibodies; somatostatin; protease inhibitors; adrenocorticotropin, gonadotropin releasing hormone; oxytocin; leutinizing-hormone-releasing-hormone; follicle stimulating hormone; glucocerebrosidase; thrombopoietin; filgrastim; prostaglandins; cyclosporin; vasopressin; cromolyn sodium; vancomycin; desferrioxamine; bisphosphonates; parathyroid hormone; anti-migraine agents; glucagon-like peptide 1 (GLP-I); antimicrobials; vitamins; analogs, fragments, mimetics or polyethylene glycol (PEG)-modified derivatives of these compounds; or any combination thereof.

45. The pharmaceutical formulation of any of claims 12-38, wherein the active agent is insulin.

46. The pharmaceutical formulation of any of claims 12-38, wherein the active agent is heparin.

47. The pharmaceutical formulation of any of claims 12-38, wherein the active agent is unfractionated heparin.

48. The pharmaceutical formulation of any of claims 12-38, wherein the active agent is low molecular weight heparin.

49. The pharmaceutical formulation of any of claims 12-48, wherein the particles have an enteric coating.

50. A solid dosage unit form comprising the pharmaceutical formulation of any of claims 12-49.

51. The solid dosage unit form of claim 50, further comprising a disintegrant.

52. The solid dosage unit form of claim 51, wherein the disintegrant is a super disintegrant.

53. The solid dosage unit form of claim 52, wherein the super disintegrant is sodium starch glycolate or croscarmellose sodium.

54. The solid dosage unit form of claim 52, wherein the super disintegrant is an extra particle super disintegrant.

55. The solid dosage unit form of any of claims 50-54, wherein the solid dosage unit form is in the form of a tablet.

56. The solid dosage unit form of any of claims 50-54, wherein the solid dosage unit form is in the form of a capsule.

57. A method of treating diabetes in a mammal in need thereof, comprising administering to the animal a therapeutic effective amount of a pharmaceutical formulation of any of claims 12-49.

58. The method of claim 57, wherein the delivery agent compound is 4-[(2- hydroxy-4-chloro-benzoyl)-amino]butanoic acid or a pharmaceutically acceptable salt thereof.

59. A method of treating impaired glucose tolerance, early stage diabetes, or late stage diabetes or achieving glucose homeostasis in humans, comprising administering a therapeutic effective amount of a pharmaceutical formulation of claim 12-49.

60. The method of claim 59, wherein the method comprises administering the pharmaceutical formulation on a chronic basis.

61. A method of treating a human diabetic patient comprising orally administering to the human diabetic patient on a chronic basis a therapeutic effective amount of a pharmaceutical formulation of any of claims 12-49.

62. A method of treating diabetes and reducing the incidence of systemic hyperinsulinemia associated with chronic dosing of insulin in a patient in need thereof comprising orally administering on a chronic basis to the patient a therapeutic effective amount of a pharmaceutical formulation of any of claims 12-49.

63., A solid dosage form comprising

(a) a therapeutically effective amount of insulin; and

(b) a delivery agent; wherein the solid dosage form has a disintegration time of at least 60 seconds when administered orally.

64. The solid dosage form of claim 63, further comprising an enteric coating.

65. The solid dosage form of claim 63, wherein the solid dosage form is a surface eroding formulation.

66. The solid dosage form of any of claims 63-65, further comprising enzyme inhibiting agents.

67. The solid dosage form of any of claims 63-66, wherein the solid dosage form comprises particles having a median particle size of less than 999 micrometers.

68. A solid dosage form comprising

(a) a therapeutically effective amount of insulin; and

(b) a delivery agent; wherein the solid dosage form does not substantially disintegrate or dissolve in the stomach but will disintegrate or dissolve in the small intestine.

69. The solid dosage form of claim 68, further comprising an enteric coating.

70. The solid dosage form of claim 68, wherein the solid dosage form is a surface eroding formulation.

71. The solid dosage form of any of claims 68-70, further comprising enzyme inhibiting agents.

72. The solid dosage form of any of claims 68-71, wherein the solid dosage form comprises particles having a median particle size of less than 999 micrometers.

73. A method of treating diabetes in a mammal in need thereof, comprising administering to the animal a therapeutic effective amount of a solid dosage form comprising the solid dosage form of any of claims 63-72.

74. A method of treating impaired glucose tolerance, early stage diabetes, or late stage diabetes or achieving glucose homeostasis in humans, comprising administering a therapeutic effective amount of the solid dosage form of any of claims 63-72.

75. The method of claim 74, wherein the method comprises administering the pharmaceutical composition on a chronic basis.

76. A method of treating a human diabetic patient comprising orally administering to the human diabetic patient on a chronic basis a therapeutic effective amount of a solid dosage form of any of claims 63-72.

77. A method of treating diabetes and reducing the instance of systemic hyperinsulinemia associated with chronic dosing of insulin in a patient in need thereof comprising orally administering on a chronic basis to the patient a therapeutic effective amount of a solid dosage form of any of claims 63-72.